Increased biliary group II phospholipase A2 and altered gallbladder bile in patients with multiple cholesterol stones

GASTROENTEROLOGY 1997;112:2036–2047

Increased Biliary Group II Phospholipase A2 and Altered Gallbladder Bile in Patients With Multiple Cholesterol Stones JUNICHI SHODA,* TETSUYA UEDA,‡ TADASHI IKEGAMI,* YASUSHI MATSUZAKI,* SUSUMU SATOH,§ MASAHITO KANO,* KENJI MATSUURA,x and NAOMI TANAKA* *Department of Gastroenterology, Institute of Clinical Medicine, University of Tsukuba, Ibaraki; ‡Department of Pharmaceutical Research, Mitsubishi Kagaku Bio-Clinical Laboratories Inc., Tokyo; §Department of Molecular Pharmacology, Pharmacological Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., Osaka; and xApplication Laboratory, MS Group, Analytical Instruments, Technical and Engineering Division, JEOL Ltd., Tokyo, Japan

Background & Aims: Multiple cholesterol stones are associated with more biliary complications and show more rapid cholesterol nucleation than solitary stones. Group II phospholipase A2 (PLA2-II) may play a critical role in the process of mucosal inflammation, which in turn may produce pronucleating agents. PLA2-II concentrations in gallbladders and gallbladder bile from patients with different types of gallstone disease were assayed to correlate PLA2-II with alterations in biliary composition. Methods: PLA2-II protein concentrations were assayed immunoradiometrically using monoclonal antibodies against human splenic PLA2-II. Results: Immunoreactive PLA2-II levels in gallbladder bile were significantly higher in patients with multiple cholesterol stones (68.2 { 6.3 ng/dL, mean { SEM; n Å 24) than in those with solitary stones (24.9 { 2.8; n Å 20; P õ 0.01), those with multiple pigment stones (24.2 { 3.7; n Å 18; P õ 0.01), or control subjects (13.4 { 1.7; n Å 19; P õ 0.01). Increased biliary immunoreactive PLA2-II levels in multiple cholesterol stones were associated with a concomitant increase in the lysophosphatidylcholine to phosphatidylcholine ratio; free arachidonate, protein, and hexosamine concentrations; and gallbladder bile viscosity. The gallbladders showed an increased PLA2-II protein mass and steadystate messenger RNA levels, which was associated with increased prostaglandin E2 levels. Conclusions: Increased biliary PLA2-II may be of pathogenetic importance in multiple cholesterol stones, probably through potentiating gallbladder mucosal inflammation with associated biliary alterations favoring cholesterol crystal formation.

T

he multifactorial nature of cholesterol gallstone formation requires hepatic secretion of cholesterol supersaturated bile, nucleation of cholesterol monohydrate crystals in the gallbladder, and subsequent growth and agglomeration of these crystals to form gallstones.1 A considerable body of literature has suggested that patients with multiple cholesterol stones (CSs), compared with those with solitary CSs, may have a different form / 5e1d$$0043

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of the gallbladder disease. Patients with multiple CSs reportedly have a higher recurrence rate after successful medical dissolution,2 exhibit much more rapid cholesterol nucleation times,3 and are more frequently accompanied by biliary complications such as acute cholecystitis and gallbladder cancer4 than those with solitary CSs. Mucosal inflammation of the gallbladder, common in cholesterol gallstone disease, may be of pathogenetic importance in producing gallbladder-derived pronucleating agents, especially because cholesterol nucleation is such a crucial step in cholesterol gallstone formation.5 Inflammation-induced gallbladder mucin has also been cited as a potent nucleating factor.6,7 These conditions may reflect more enhanced mucosal inflammation in the gallbladders of patients with multiple CSs than in those with solitary CSs. Recently, interest has been focused on the role played by group II phospholipase A2 (PLA2-II), which has a molecular weight of 14 kilodaltons, as an inflammatory mediator. A large amount of PLA2 -II has been found in synovial fluid of patients with rheumatoid arthritis.8 In addition, intra-articular injection of purified PLA2 -II induces dose-dependent acute inflammatory changes in the synovial tissue.9 Increased PLA2 activity found in the ileal and colonic mucosa of Crohn’s disease,10,11 mainly attributed to PLA2 immunochemically related to PLA2II, suggests involvement of PLA2-II in the pathogenesis of intestinal inflammation. Also, interleukin 1 has been shown to increase PLA2 activity, PLA2 -II messenger RNA (mRNA) levels, and prostaglandin (PG) E2 synthesis in rabbit chondrocytes.12,13 These observations support Abbreviations used in this paper: cPLA2 , cytosolic phospholipase A2 ; CS, cholesterol stone; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; HPLC, high-performance liquid chromatography; IR, immunoreactive; LPC, lysophosphatidylcholine; PC, phosphatidylcholine; PLA2-II, group II phospholipase A2 ; PS, pigment stone; RT-PCR, reverse-transcription polymerase chain reaction. q 1997 by the American Gastroenterological Association 0016-5085/97/$3.00

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the hypothesis that directly or indirectly, PLA2 -II or its derivatives may play a critical role in the pathogenesis of inflammatory responses.14 Extracellular activity of PLA2 -II at inflamed sites15 and in circulating blood16 may enable gallbladder-derived PLA2 -II to be secreted into gallbladder bile, where it could potentiate alterations in biliary composition favoring cholesterol crystal formation. To elucidate the role of PLA2 -II in the pathogenesis of multiple CSs, we determined PLA2 -II concentrations in gallbladders and gallbladder bile of patients with different types of gallstone disease, correlating these with altered biliary composition and with gallbladder PG synthesis.

Materials and Methods Nineteen normolipidemic stone-free subjects and 62 patients with gallstones (20 with solitary CSs, 24 with multiple CSs, and 18 with multiple black pigment stones [PSs]) were included in this study. The presence of gallstones was confirmed by preoperative ultrasonography and interoperative palpation. In all subjects, the gallbladders were well-functioning as determined by preoperative drip infusion cholangiography and a total biliary lipid concentration of ú5 g/dL. All patients with gallstones were undergoing elective cholecystectomies, and none of the patients had shown any clinical or laboratory evidence of acute cholecystitis during the 4 months before the procedures. Patients with nonvisualized gallbladders were excluded. All subjects were admitted to hospital at least a week before operations and kept on a regular diet (2125 kcal/day, containing 82.2 g of protein, 56.7 g of lipids, and 315 g of carbohydrates). No control subjects or patients with gallstones had any evidence of hepatic, intestinal, or renal disease, cholangitis, diabetes mellitus, or thyroid dysfunction. None had taken antibiotics, lipid-lowering drugs, bile acids, or any hormones within the 4 weeks preceding the sample collections. Most control subjects were undergoing surgery for small adenomatous polyps of the gallbladder (õ20 mm in diameter); remaining controls had early gastric cancer. Age, relative body weight, and serum lipid concentrations did not differ significantly among the four groups. We obtained each patient’s informed consent, and conducted the study in accordance with the ethical standards of the Helsinki Declaration.

Gallstones The gallstones obtained at surgery were classified by visual inspection and infrared analysis.17 The number and mean diameter of the gallstones obtained at surgery were recorded and an estimate of total gallstone volume was obtained using the formula (4/3) p r3 1 n, in which r is the radius of the average stone in a particular gallbladder and n is the number of stones. Gallstones were washed with distilled water, dried to a constant weight at room temperature, and ground to powder. Lipids were extracted into ethanol from the powder by a method described previously.18 Cholesterol concentration

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Bile Gallbladder bile was aspirated from the gallbladder using a sterile needle and syringe as described previously,20 with particular care taken to avoid the effects of stratification21 and contamination with blood. Immediately after the collection of bile, phenylmethylsulfonyl fluoride, an antiprotease, was added to the bile specimens to give a final concentration of about 1 mmol/L phenylmethylsulfonyl fluoride. The specimens were stored at 0807C until analyses. Aliquots of the specimens were stored in chloroform and methanol (2:1, vol/vol) at 0207C for lipid analyses.

Gallbladders

Subjects

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was determined using Cholesterol E-Test Wako. CSs were defined as stones with cholesterol constituting more than 70% of total weight.19

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Surgically resected gallbladders were opened and then sent for histological examination. Histological examination of the removed gallbladders showed either no or slight chronic inflammation. Small specimens of the gallbladder tissue were immediately washed with distilled water and frozen in liquid nitrogen. The gallbladder specimens were stored at 0807C until analysis.

Chemicals and Enzymes Aminopropyl-bonded silica (Bond-Elut NH2 100 mg) cartridges were obtained from Varian Co. (Harbor City, CA). Colorimetric enzymatic assay kits (Cholesterol E-Test Wako, Phospholipid C-Test Wako, and Total Bile Acids-Test Wako) were obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Fluorescamine, borax, and boric acid were obtained from Sigma Chemical Co. (St. Louis, MO). Trichloroacetic acid was obtained from Fisher Scientific Co. (Pittsburgh, PA). Bradford reagent was obtained from Bio-Rad (Richmond, CA). Total RNA extraction solution (Trizol reagent), random primers, and Moloney murine leukemia virus reverse transcriptase were obtained from GIBCO-BRL (Gaithersburg, MD). Taq DNA polymerase was obtained from Boehringer Mannheim (Mannheim, Germany). All other chemicals were reagent grade. Water was double distilled and deionized with a super Q water system (Millipore Corp., Bedford, MA).

PLA2-II Assay Radioimmunoassay. The protein masses of PLA2 -II levels in gallbladder bile (ng/dL) and gallbladders (ng/ mgrprotein) were immunoradiometrically assayed as described previously,22 with a minor modification using a combination of two monoclonal antibodies against purified human splenic PLA2 -II, as reported recently.23 Assay kits were kindly supplied from Pharmaceuticals Research & Development Division, Shionogi & Co. Ltd. (Osaka, Japan). Fifty to 100 mL of bile and 25 mL of supernatants of gallbladder homogenates (100– 400 mg of protein) were used for an assay. All assays were performed in triplicate. Gallbladder homogenate was obtained by homogenizing the frozen tissues in 50 mmol/L Tris-HCl

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(pH 7.4), containing 100 mmol/L NaCl and 1 mmol/L ethylenediaminetetraacetic acid (EDTA). The supernatant was obtained by centrifuging the homogenate at 15,000g for 15 minutes at 47C. Protein concentration in the supernatant was measured by the method described by Lowry et al.24 PLA2 enzyme activity. PLA2 activity was determined as described previously.25 Briefly, the assay was performed using 0.8 mmol/L 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol as a substrate in the presence of 5 mmol/L cholate. Fatty acids released by PLA2 were labeled with 9-anthryldiazomethane, and the derivatized fatty acids were separated by high-performance liquid chromatography (HPLC). The oleic acid was quantitated with marganic acid as an internal standard. Immunoblot analysis. The supernatant of the gallbladder homogenate was diluted threefold with Tris-HCl buffer and then applied to a heparin Sepharose column (15 1 60 mm). After washing with Tris-HCl buffer, the elution of PLA2 -II was performed with 50 mmol/L Tris-HCl (pH 7.4), containing 1 mol/L NaCl and 1 mmol/L EDTA. The fractions containing PLA2 -II were combined, then a concentrated PLA2 II fraction was obtained by freeze-drying the combined fractions after dialysis. Immunoblot analysis was performed on a 15% sodium dodecyl sulfate–polyacrylamide gel under nonreducing conditions. The proteins in gel were electrophoretically transferred onto Immobilon membranes (Millipore Corp., Bedford, MA). The membrane was incubated with anti-human splenic PLA2 -II monoclonal antibodies (a gift from Pharmaceuticals Research & Development Division, Shionogi & Co., Ltd.). The resulting antigen-antibody complex was detected using an ECL kit (Amersham, London, England). RNA isolation and reverse-transcription polymerase chain reaction. Total RNA was isolated from the gallbladder

specimens using Trizol reagent by the modified method as described by Chomczynski and Sacchi.26 First-strand complementary DNAs (cDNAs) were synthesized from total RNA with Moloney murine leukemia virus reverse transcriptase by the random primer method. Polymerase chain reaction (PCR) was performed using 50 pmol of each primer, 200 ng of cDNA template, 1.25 units of Taq DNA polymerase, 200 nmol/L deoxynucleoside triphosphate, and 11 reaction buffer (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl2 , and 0.01% gelatin) in a 50 mL reaction volume using DNA Thermal Cycler (model PJ 2000; Applied Biosystems Inc., Foster City, CA). PCR27 was subjected to each cycle (PLA2-II, 40; cytosolic PLA2[cPLA2], 40; glyceraldehyde-3phosphate dehydrogenase [G3PDH], 22) at 947C for 1 minute, at 557C for 2 minutes, and at 727C for 2 minutes. Aliquots of the reaction mixture were electrophoresed on a 2% agarose gel. In the experiments involving quantitative assessment, the amounts of fluorescence intensity were measured by FluorImager (Molecular Dynamics, Sunnyvale, CA). The data are expressed relative to the amounts of G3PDH mRNA present in each specimen. PCR primers were designed from cDNA sequences for human PLA2 -II28 and cPLA2 ,29 and then synthesized by an Applied Biosystems DNA synthesizer (model 392; Applied Biosystems Inc.) as follows: G3PDH, sense 5*-

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GAACGGGAAGCTCACTGGCATGGC-3*, antisense 5*TGAGGTCCACCACCCTGTTGCTG-3*; PLA2-II , sense 5*-ACCATGAAGACCCTCCTACTG-3*, antisense 5*-GAAGAGGGGACTCAGCAACG-3*; and cPLA2 , sense 5*-CGGTAGTGGTGTTACGTGCCACC-3*, antisense 5*-GGCTACCACAGGCACATCACGTG-3*. Nucleation time. The fresh bile specimens were examined for the presence of cholesterol monohydrate crystals and ultracentrifuged at 377C for 2 hours at 105g in a Beckman L50 ultracentrifuge (Beckman Instruments, Fullerton, CA) to obtain crystal-free bile as described by Holan et al.30 The crystal-free bile specimens were placed into sterile glass tubes at 377C in the dark under nitrogen. An aliquot was examined immediately to confirm the absence of cholesterol monohydrate crystals and subsequently examined daily for their appearance at 377C under a polarized microscope (Nikon XTP-II, Tokyo, Japan). All specimens were observed over 21 days. When cholesterol crystals did not appear during the observation period, the nucleation time was recorded as 21 days. The sterility of all bile specimens studied was verified by bacteriological examination. Biliary lipids, protein, and hexosamine. The concentrations of cholesterol and phospholipids were determined by enzymatic methods using Cholesterol E-Test Wako31 and Phospholipid C-Test Wako,32 respectively, after eliminating the bilirubin using a Bond-Elut NH2 cartridge.33 The total bile acid concentration was determined by an enzymatic method using Total Bile Acids-Test Wako.34 The cholesterol saturation of bile was calculated according to the critical tables for cholesterol saturation based on the total lipid concentration.35 The fatty acid pattern of the free fatty acid fraction of bile, separated by thin-layer chromatography,36 and that of the phospholipid fraction, extracted by a Bond-Elut NH2 cartridge,37 were determined by gas-liquid chromatography as described previously.38 Phospholipids in gallbladder bile specimens were extracted by a Bond-Elut NH2 cartridge as described previously,37 and the concentrations of biliary phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) were determined using HPLCmass spectrometry39 (MS, JEOL LX2000 mass spectrometer with Frit-FAB LC/MS interface, JEOL Ltd., Tokyo, Japan; HPLC, Hewlett-Packard 1090L, Hewlett-Packard Co., Avondale, PA). Quantitation of PC and LPC was performed using the most intense fragment ion at m/z 184, derived from the choline part of 1-palmitoyl-2-oleyl-phosphatidylcholine and specific for PC species, and using the intense protonated molecular ion at m/z 496, derived from 1-palmitoyl lysophosphatidylcholine and specific for LPC species, respectively. Good recoveries of PC (Ç91%) and LPC (Ç84%) were obtained by this procedure. Total biliary protein was measured by the fluorescamine method40 as applied to bile specimens.41 Briefly, bile was diluted 10-fold with water. Protein was precipitated from 0.5 mL of the diluted specimen with 0.5 mL of 20% trichloroacetic acid. The solution was then incubated overnight and centrifuged at 15,000g for 10 minutes at 47C. The resulting pellet

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Table 1. Data for Patients With Gallstones

Subjects

n

Sex (M/F)

Controls Patients with Solitary CSs Multiple CSs Multiple PSs

19

10/9

20 24 18

4/16 8/16 6/12

Gallstone volume (mL)

Cholesterol content (% by dry wt )

—

—

3.8 { 0.4a 4.8 { 0.5 3.2 { 0.7

95.8 { 2.2 90.2 { 2.6 10.9 { 0.9b

NOTE. All multiple PSs were classified as black PSs by visual inspection and infrared analysis. a Values are given as means { SEM. b Significantly different from patients with solitary CSs (P õ 0.01) and patients with multiple CSs (P õ 0.01).

was washed with 1 mL of ether-ethanol (3:1, vol/vol) for delipidation and then recentrifuged. The final pellet was dissolved in 0.5 mL of borate buffer (pH 9.0) and incubated overnight at 47C. After the addition of 1 mL of borate buffer and 0.5 mL of fluorescamine solution (0.02% in acetone) to the solution, the fluorescence was measured at an emission wavelength of 480 nm and excitation wavelength of 390 nm with a fluorescence spectrophotometer (Shimadzu RF-1500, Kyoto, Japan). Hexosamine concentration in bile was determined as described previously42 with minor modifications. Gallbladder bile (0.1 mL) was diluted with 5 mL of 95% aqueous ethanol and then mixed. After centrifugation at 500g for 15 minutes, the pellet was obtained by aspirating the supernatant. This step was repeated twice. The resulting pellets were dissolved in 2 mL of 3N hydrochloric acid and then refluxed at 1007C for 4 hours. After neutralization with 3N sodium hydroxide, the reaction mixture with 1 mL of acetylacetone reagent was placed again at 1007C for 15 minutes. After adding 1 mL of p-dimethylaminobenzaldehyde (Ehrlich’s reagent) and diluting to 10 mL with 95% ethanol, the reaction mixture was incubated at 457C for 30 minutes, then a colorimetric assay was performed at a wavelength of 530 nm. PGE2 assay. Frozen gallbladder tissue was transferred in ice-cold buffer (pH 8.4) and then homogenized. The supernatant was stored at 0207C. Aliquots were assayed by highly specific radioimmunoassay43 (anti-PGE2 antibody, Amersham) for PGE2 in duplicate and at two dilutions. The protein contents in the supernatant were measured by the method described by Lowry et al.24 The final results are expressed as pg PGE2 /mgrprotein. Viscosity of gallbladder bile. Viscosity of bile (mPa/s) was measured using a capillary viscometer (Full Automatic Capillary Viscometer Model hma 200; Yokohama Hamax Co. Ltd., Yokohama, Japan). The fixed volume of bile specimens is drawn horizontally through the capillary resistance element under negative pressure according to needs by an air pump and linear valve, and then the flow rate is measured precisely. The flow rate of specimens along a capillary length and radius is related to the pressure by the Hagen–Poiseuille relation as follows: h Å pR4P/8LQ, where h is viscosity, R is capillary radius, P is pressure, L is capillary length, and Q is flow rate. Statistics. Values are given as means { SEM. The

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statistical significance of differences in the values between different groups was evaluated with the Mann–Whitney U test (two-tailed test). Correlation was tested by calculating Spearman’s rank correlation coefficient, r (two-tailed test). A P value of õ0.05 was defined as statistically significant.

Results Gallstone Analysis The gallstone volume in patients with multiple CSs was larger than that in patients with solitary CSs, and the gallstone cholesterol content in solitary CSs was greater than that in multiple CSs; neither difference was significant (Table 1). PLA2-II in Gallbladder Bile and Gallbladder Immunoreactive (IR) PLA2-II levels in gallbladder bile were increased significantly in gallstone patients, irrespective of stone category, compared with control subjects (Figure 1). Furthermore, biliary IR PLA2-II levels were increased significantly in patients with multiple CSs compared with those with solitary CSs. As shown in Figure 2, the immunoblot analysis showed that the gallbladder bile of a patient with multiple CSs included an enzyme protein mass immunochemically cross-reactive with antibodies raised against purified human splenic PLA2 -II. When enzyme activities of PLA2 were compared with the IR PLA2 -II levels in gallbladder bile, IR PLA2 -II levels were correlated with PLA2 enzymatic activity (Figure 3; n Å 44; r Å 0.66; P Å 0.0001). The biliary PLA2 activity was 1.4 { 0.2 nmolrmin01rmL01 (mean { SEM) for 8 of the control subjects, 1.5 { 0.1 for 12 of the patients with solitary CSs, 2.8 { 0.7 for 16 of those with multiple CSs, and 1.9 { 0.6 for 8 of

Figure 1. IR PLA2 -II levels in gallbladder bile from control subjects and patients with different types of gallstone disease. Biliary IR PLA2 -II levels were 13.4 { 1.7 ng/dL (mean { SEM) for control subjects, 24.9 { 2.8 for patients with solitary CSs, 68.2 { 6.3 for patients with multiple CSs, and 24.2 { 3.7 for patients with multiple PSs.

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Figure 2. Immunochemical relationship between biliary and gallbladder PLA2s and PLA2 -II purified from human spleens. Lane 1, human splenic PLA2-II; lane 2, immunoblot analysis of gallbladder bile and gallbladder homogenates with anti-human PLA2 -II antibody. The positions of molecular weight markers running in adjacent lanes were determined by staining the nitrocellulose filter with Ponceau S before blocking the filter.

those with multiple PSs. Compared with control subjects, patients with multiple CSs had a twofold increase in PLA2 activity. However, the difference was not significant due to the wide distribution of PLA2 activity between control subjects and patients with multiple CSs (Figure 3). Addition of EDTA at a concentration of 5 mmol/L in the bile was found to abolish most (72.1%– 98.5%) of this PLA2 activity, thereby indicating the contribution of calcium-dependent PLA2 to biliary PLA2 activity. Consistent with biliary IR PLA2-II levels, IR PLA2II levels in gallbladders were significantly increased in patients with multiple CSs (4.1 { 0.6 ng/mgrprotein, means { SEM) compared with those with solitary CSs (2.1 { 0.3, P õ 0.05) or control subjects (1.3 { 0.3, P õ 0.05) as shown in Figure 4. The immunoblot analysis of gallbladder homogenates also showed that the gallbladder of a patient with multiple CSs included an enzyme protein mass immunochemically cross-reactive with anti-human splenic PLA2-II antibodies (Figure 2). There was a significant linear correlation between IR PLA2-II levels in the gallbladder bile and the gallbladder (n Å 31; r Å 0.49; P Å 0.004). In association with the increased IR PLA2-II levels in the gallbladders, the gallbladder PGE2 concentrations were elevated in patients with solitary CSs (288.8 { 62.9 pg/mgrprotein, mean { SEM), those with multiple CSs (780.4 { 131.1), and those with multiple PSs (345.8 { 89.3) compared with control subjects (98.0 { 19.2) (Figure 4). The PGE2 concentrations were significantly higher in patients with multiple CSs than in patients / 5e1d$$0043

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with CSs (P õ 0.01), those with multiple PSs (P õ 0.05), or control subjects (P õ 0.01). Reflecting the stimulated arachidonate metabolism by the increased IR PLA2-II levels in the gallbladders, gallbladder IR PLA2II levels were correlated closely with the PGE2 concentrations (n Å 46; r Å 0.85; P Å 0.0001). To determine whether the increases in biliary and gallbladder IR PLA2-II levels are attributable to the production and secretion of PLA2-II after the mRNA production, the gallbladder steady-state mRNA was determined by reverse-transcription (RT)-PCR. Figure 5 shows the PCR-assisted amplification of gallbladder PLA2 -II and cPLA2 mRNA levels in different types of gallstone diseases. The abundance of PLA2 -II mRNA (Figures 5A and 6) was higher in the gallbladders of patients with multiple CSs (2.4 { 0.5, mean { SEM) than in those of control subjects (0.9 { 0.1), patients with solitary CSs (1.4 { 0.1, P õ 0.05), or multiple PSs (1.2 { 0.1, P õ 0.05). In contrast to PLA2-II, the abundance of cPLA2 (Figure 5B), which preferentially hydrolyzes phospholipid molecular species containing arachidonate, did not differ in patients with multiple CSs, compared with that of patients with solitary CSs or multiple PSs. Biliary Lipid Composition The total lipid, cholesterol, phospholipid, and total bile acid concentrations and the cholesterol saturation index (CSI; solitary CSs, 1.32 { 0.07, mean { SEM; multiple CSs, 1.44 { 0.09) in gallbladder bile were similar between patients with solitary CSs and those with multiple CSs. Cholesterol monohydrate crystals were observed in 75% of patients with solitary CSs and in 92%

Figure 3. Correlation between biliary IR PLA2 -II and PLA2 enzymatic activity in control subjects and patients with different types of gallstone disease. s, Control subject; , patients with solitary CSs; , patients with multiple CSs; and l, patients with multiple PSs; n Å 44, r Å 0.66, P Å 0.0001.

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Figure 4. IR PLA2 -II and PGE2 levels in gallbladders from control subjects and patients with different types of gallstone disease. IR PLA2 -II levels in gallbladders were 1.3 { 0.3 ng/mgrprotein (mean { SEM) for control subjects, 2.1 { 0.3 for patients with solitary CSs, 4.1 { 0.6 for patients with multiple CSs, and 2.8 { 0.5 for patients with multiple PSs. The PGE2 levels were 98.0 { 19.2 pg/mgrprotein (mean { SEM) for control subjects, 288.8 { 62.9 for patients with solitary CSs, 780.4 { 131.1 for patients with multiple CSs, and 345.8 { 89.3 for patients with multiple PSs.

of those with multiple CSs. However, the cholesterol nucleation time was significantly faster in the bile of patients with multiple CSs (2.9 { 0.5 days, mean { SEM; from less than 1 day to 9 days; median, 2 days) than in the bile of those with solitary CSs (4.7 { 0.7, P õ 0.05; from less than 1 day to 14 days; median, 3 days). The concentrations of total lipid, cholesterol, phospholipid, bile acid, and the CSI values in patients with multiple PSs were similar to those in control subjects. Cholesterol monohydrate crystals were absent in patients with multiple PSs and control subjects, and the nucleation time did not differ between patients with multiple PSs and control subjects (Table 2). In proportion to the increased IR PLA2-II levels, the LPC to PC ratio and free arachidonate concentration were significantly increased in gallbladder bile of patients with multiple CSs (6.3 { 1.1 1 1002, mean { SEM; 28.7 { 5.6 mg/mL) compared with the corresponding data of patients with solitary CSs (3.1 { 0.4 1 1002, P õ 0.05; 10.3 { 1.5, P õ 0.01) or control subjects (1.5 { 0.1 1 1002, P õ 0.01; 7.7 { 1.4, P õ 0.01). Biliary IR PLA2-II levels were correlated closely with the LPC to PC ratio (n Å 54; r Å 0.64; P Å 0.0001) and the free arachidonate concentration (n Å 54; r Å 0.74; P Å 0.0001) as shown in Figure 7A. The total protein concentration in gallbladder bile was significantly increased in patients with solitary or multiple CSs compared with control subjects and patients with multiple PSs. In a comparison between soli/ 5e1d$$0043

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Figure 5. RT-PCR–assisted amplifications of (A ) PLA2 -II and (B ) cPLA2 mRNAs in gallbladders of patients with different types of gallstone disease. The abundance of G3PDH mRNA was determined as an internal standard.

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Figure 6. Relative steady-state levels for gallbladder PLA2 -II mRNA in control subjects and patients with different types of gallstone disease. The levels were 0.9 { 0.1 (mean { SEM) for control subjects, 1.4 { 0.1 for patients with solitary CSs, 2.4 { 0.5 for patients with multiple CSs, and 1.2 { 0.1 for patients with multiple PSs.

tary CSs and multiple CSs, the total biliary protein concentration was significantly higher in patients with multiple CSs (4.0 { 0.4 mg/mL, mean { SEM) than in those with solitary CSs (2.9 { 0.2, P õ 0.05). When biliary IR PLA2-II levels were compared with the total protein concentration in gallbladder bile, there was a significant correlation (n Å 81; r Å 0.77; P Å 0.0001) as shown in Figure 7B.

In addition to the measurement of total protein concentration, the hexosamine concentration in gallbladder bile was determined as an indicator of biliary mucus glycoprotein amounts.44 The hexosamine concentration was significantly higher in patients with gallstones (solitary CSs, 0.89 { 0.10 mg/mL, mean { SEM, P õ 0.01; multiple CSs, 1.62 { 0.17, P õ 0.01; multiple PSs, 1.06 { 0.12, P õ 0.01) than in control subjects (0.48 { 0.06). In a comparison between solitary CSs and multiple CSs, biliary hexosamine concentration was significantly higher in patients with multiple CSs than in those with solitary CSs (P õ 0.01). When biliary IR PLA2 -II levels were compared with the hexosamine concentration in gallbladder bile, there was a significant correlation (n Å 65; r Å 0.80; P Å 0.0001) as shown in Figure 7B. The viscosity of gallbladder bile from patients with gallstones was measured at 377C using a microviscometer. Biliary viscosity was higher in patients with gallstones (solitary CSs, 3.3 { 0.2 mPars, mean { SEM; multiple CSs, 6.1 { 1.1; multiple PSs, 3.5 { 0.6) than in control subjects (2.4 { 0.2). The biliary viscosity was significantly higher in patients with multiple CSs than in those with solitary CSs (P õ 0.01) or control subjects (P õ 0.01). In addition, the biliary viscosity at 377C was correlated closely with total protein concentration (n Å 66; r Å 0.66; P Å 0.0001), hexosamine concentration (n Å 61; r Å 0.74; P Å 0.0001), and IR PLA2-II levels in gallbladder bile (n Å 67; r Å 0.71; P Å 0.0001) as shown in Figure 7B.

Table 2. Comparison of Gallbladder Bile of Patients With CSs, Patients With Multiple PSs, and Control Subjects Gallstones

Total lipid concentration (g/dL) Cholesterol (mmol/L) Phospholipids (mmol/L) Total bile acids (mmol/L) Cholesterol saturation index Cholesterol crystal (%) Nucleation time (day ) Free arachidonate (mg/mL) LPC/PC ratio 1 102 Total protein concentration (mg/mL) Hexosamine concentration (mg/mL) Viscosity (mPa/s)

Controls (n Å 19)

Solitary cholesterol (n Å 20)

Multiple cholesterol (n Å 24)

Multiple pigment (n Å 18)

14.2 { 1.1 14.8 { 1.3 55.7 { 4.1 189.1 { 14.1 0.77 { 0.06 0/19 (0) 15.9 { 1.4 (19) 7.7 { 1.4 1.5 { 0.1 1.8 { 0.1 0.48 { 0.06 2.4 { 0.2

11.6 { 1.3 16.3 { 1.4 44.5 { 4.6 140.3 { 9.4b 1.32 { 0.07b 15/20 (75) 4.7 { 0.7 (3)b 10.3 { 1.5 3.1 { 0.4a 2.9 { 0.2b 0.89 { 0.10b 3.3 { 0.2

10.8 { 1.4 15.4 { 1.2 42.3 { 3.9a 126.9 { 9.5b 1.44 { 0.09b 22/24 (92) 2.9 { 0.5 (2)b,c 28.7 { 5.6b,d 6.3 { 1.1b,c 4.0 { 0.4b,c 1.62 { 0.17b,d 6.1 { 1.1b,d

12.8 { 1.0 11.2 { 1.5 51.3 { 2.6 169.5 { 16.2 0.89 { 0.07 0/18 (0) 14.6 { 1.3 (16) 12.3 { 3.0 2.2 { 0.4 2.0 { 0.2 1.06 { 0.12a 3.5 { 0.6b

NOTE. Values are given as means { SEM. Medians of nucleation time are shown in parentheses. The ranges of nucleation time were from 6 to 21 days in controls, less than 1 to 14 days in solitary CSs, less than 1 to 9 days in multiple CSs, and 4 to 9 days in multiple PSs, respectively. Free arachidonate concentrations and the LPC/PC ratio were determined for 15 control subjects and 39 gallstone patients (13 solitary CSs, 13 multiple CSs, and 13 multiple PSs). Hexosamine concentrations were determined for 19 control subjects and 46 gallstone patients (17 solitary CSs, 14 multiple CSs, and 15 multiple PSs). Viscosity of gallbladder bile at 377C was determined for 19 control subjects and 48 gallstone patients (18 solitary CSs, 16 multiple CSs, and 14 multiple PSs). a,b Significantly different from control subjects; a, P õ 0.05; b, P õ 0.01. c,d Significantly different from patients with solitary cholesterol stones; c, P õ 0.05; d, P õ 0.01.

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Figure 7. (A ) Correlation between the IR PLA2 -II levels and the LPC to PC ratio, and the free arachidonate (AA) concentrations in the gallbladder bile. (B ) Correlation between the IR PLA2 -II levels and the total protein concentrations, the hexosamine concentrations, and the viscosity at 377C of the gallbladder bile. s, Control subjects, , patients with solitary CSs; , patients with multiple CSs; and l, patients with multiple PSs. Shaded areas represent the distribution of control subjects.

Discussion Considerable evidence2 – 4 supports the notion that patients with multiple CSs represent a different form of cholesterol gallstone disease compared with those with solitary CSs. The more rapid cholesterol nucleation time in multiple CSs3 may reflect the effects of more intense gallbladder mucosal inflammation on alterations in biliary composition, especially in the production of gallbladder-derived pronucleating agents, including mucin. This more intense inflammation also may be associated with more frequent biliary complications in patients with multiple CSs.4 Recently, attention has been focused on the role played by PLA2 in the initiation and propagation of the inflammatory process.14,45 Increased intestinal concentrations of PGs and leukotriene B446,47 and increased PLA2 activity in ileal and colonic mucosa of patients with inflammatory bowel disease10,11 suggest that PLA2 activity is influential in the pathogenesis of intestinal inflammation. Seeking pathogenetic factors involving gallbladder mucosal inflammation that might favor formation of multiple CSs, we correlated PLA2 -II levels in gallbladders and gallbladder bile of patients with multiple CSs with physicochemical and biochemical alterations / 5e1d$$0043

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of their gallbladder bile. To our knowledge, this study provides the first direct evidence for an increased protein mass of PLA2 -II in gallbladders and gallbladder bile of patients with multiple CSs. In this study, biliary IR PLA2-II levels (reflecting PLA2 -II protein mass) were correlated with calcium-dependent PLA2 activity in gallbladder bile. As shown by the immunoblot analysis (Figure 2), the enzymes in both gallbladder and gallbladder bile were immunochemically cross-reactive with the monoclonal antibodies raised against human PLA2-II from human spleens. All specimens gave bands having the same mobility as the purified splenic PLA2-II, indicating that these enzymes have the same apparent molecular weight (14 kilodaltons) as the purified splenic PLA2-II, whereas cross-reactivities with other human PLA2 such as the pancreatic form of PLA2 or group I PLA2 were negligible (data not shown). On the other hand, the correlation between IR PLA2 -II levels and PLA2 activity in the bile was not as close as that between IR PLA2 -II levels and PLA2 enzymatic activity in sera, as reported previously.48 The results imply the presence in gallbladder bile of factors such as PLA2 activating protein,49 inhibitory protein,50 and others important in modulating PLA2 activity. However, the aboWBS-Gastro

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lition of PLA2 activity in gallbladder bile by the addition of EDTA or a specific inhibitor of human PLA2-II (data not shown) supports the conclusion that PLA2 -II may be the most critical enzyme responsible for calcium-dependent PLA2 activity in gallbladder bile. We sought to clarify the source of biliary PLA2-II of which protein mass and enzymatic activity were increased in the bile of patients with multiple CSs. It can be argued that increased biliary IR PLA2 -II levels may be due to responses to local inflammation in gallbladders of patients with gallstones. In the gallbladders of patients with multiple CSs, the expression levels of mRNA (Figure 5A) and the protein mass of PLA2 -II (Figure 4) were significantly higher than in control subjects or patients with solitary CSs. These high expression levels of PLA2 II mRNA in gallbladders with multiple CSs indicate enhanced de novo PLA2 -II synthesis in the gallbladder. However, platelets51 or various inflammatory cells14 have been known to secrete PLA2-II on stimulation. Therefore, the possibility that these cells may contribute to increased biliary PLA2-II should be given some consideration. On the other hand, the recently observed or cPLA2 , enzymes with a high molecular weight of 60–110 kilodaltons,52 preferentially hydrolyze phospholipid molecular species containing arachidonate.29 In contrast to PLA2 -II, the expression levels of cPLA2 mRNA did not differ significantly in patients with multiple CSs, compared with control subjects, those with solitary CSs, or those with multiple PSs. However, this observation does not exclude a contribution of cPLA2 in the pathogenesis of cholesterol stones. IR PLA2 -II levels in both gallbladders and gallbladder bile were significantly higher in patients with multiple CSs than in control subjects or those with solitary CSs (Figures 1 and 4). In addition, the enzyme activities of PLA2 in the bile were found to be parallel to the IR PLA2 -II levels (Figure 3). Reflecting this, biliary IR PLA2 -II levels were also correlated closely with the LPC to PC ratio and free arachidonate concentrations. An important observation was the association of altered physicochemical and biochemical alterations of gallbladder bile with increased biliary IR PLA2 -II levels. PLA2 hydrolyzes fatty acids bonded at the sn-2 position of glycerophospholipids and produces lysophospholipids.53 Accordingly, the increases in the LPC to PC ratio and free arachidonate concentration may be considered alterations attributable to the increased PLA2 activities paralleling the IR PLA2 -II levels. Thus, PLA2 -II secreted from gallbladder wall into bile may, at least in part, contribute to the liberation of arachidonate from either biliary or intramucosal arachidonyl lecithins and production of LPC. Previous reports54,55 have implicated LPC in the / 5e1d$$0043

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pathogenesis of acute mucosal inflammation of the gallbladder. In addition, the increased levels of arachidonate in gallbladder bile may enrich the gallbladder mucosa and in turn provide a substrate for gallbladder PG synthesis.56 The roles of increased biliary LPC concentrations and enhanced gallbladder PG synthesis in evolving cholecystitis were confirmed experimentally using rabbits with bile duct ligation.57 PG synthesis has been shown in human gallbladders with histological change of chronic cholecystitis,58 and its role in the pathophysiology of cholecystitis has been discussed.59 In the pathogenesis of cholesterol gallstone disease, the role of PGs in gallbladder mucin hypersecretion60,61 remains controversial.62 PGE2 has been linked to mucus gland proliferation in bile ducts using animal models treated with PGE2 .63 Accordingly, interest has focused recently on the contributions of PLA2 -II and cPLA2 activities to the liberation of arachidonate and to PG synthesis, especially that of PGE2 . Synthesis and secretion of PLA2 -II triggered by proinflammatory cytokines64 and a marked increase in PGE2 synthesis paralleling PLA2 -II release have been shown in in vitro experiments.65 Consistent with these observations, the gallbladder concentrations of PGE2 were more pronounced in patients with solitary or multiple CSs than in control subjects, and the PGE2 concentrations were significantly higher in patients with multiple CSs than in those with solitary CSs. The correlation between IR PLA2 -II levels and PGE2 concentrations indicates increased PGE2 synthesis paralleling PLA2 -II production in the gallbladder. Also, a concomitant increase in hexosamine concentration and biliary viscosity in patients with multiple CSs may link increased PGE2 synthesis with enhanced gallbladder mucin secretion. Increased levels of total biliary protein and glycoprotein have been associated with more rapid cholesterol nucleation.66 Consistent with a previous report3 and recent work in our laboratory,67 we also observed more rapid cholesterol nucleation in patients with multiple CSs than in those with solitary CSs, and confirmed the association of rapid nucleation with increased total biliary protein and hexosamine concentrations. Considering the correlations of IR PLA2 -II levels with total protein and hexosamine concentrations, increased PLA2 -II may heighten gallbladder mucosal inflammation (cholecystitis), which in turn causes either increased mucosal permeability with a subsequent leakage of proteins into the bile11,66 (presumably originating from the blood or gallbladder wall), or enhanced glycoprotein secretion possibly mediated by PGs.60,61 Another interesting finding was the high gallbladder bile viscosity at 377C in patients with multiple CSs paralWBS-Gastro

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Figure 8. Putative scheme for the cascade of PLA2 -II–mediated inflammatory responses in both gallbladder wall and gallbladder bile in cholesterol gallstone disease.

leled by increased biliary IR PLA2 -II levels. The higher levels of these biliary protein constituents may be causatively related to increased viscosity. Cholesterol supersaturated bile with heightened viscosity allows prolonged stasis of cholesterol-enriched vesicles and crystals and ensures multicentric growth of crystals into multiple stones in the gallbladder. One hypothesis of the cascade of PLA2 -II-mediated inflammatory responses in the gallbladders with cholesterol stones is illustrated in Figure 8. Induction of PLA2 II synthesis in response to proinflammatory stimuli may occur in the gallbladder mucosa. The synthesized PLA2 II is secreted into the lumen and then hydrolyzes arachidonyl-phosphatidylcholine in the biliary mixed micelles and vesicles or membranous arachidonyl-phosphatidylcholine on the mucosa, yielding lysophosphatidylcholine and free arachidonate. The bile becomes enriched with free arachidonate, which in turn enriches the mucosa with arachidonate. This process is repeated by the cycles of gallbladder filling and emptying of bile rich in arachidonyl-phosphatidylcholine. Enrichment of the mucosa with arachidonate provides a substrate for PG synthesis (i.e., PGE2 ). The generated PGs mediate not only propagation of mucosal inflammation but also mucin hypersecretion. These pathological conditions lead to the formation of bile with higher protein (presumably of the blood or gallbladder wall origin) and mucin concentrations (both of the molecules generating higher viscosity of the bile), which in turn may underlie rapid nucleation of cholesterol crystals. However, the difference in the PLA2 -II-mediated inflammatory responses between solitary and multiple cholesterol stones is not yet fully understood. Cholesterol / 5e1d$$0043

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crystals reportedly act as proinflammatory agents and induce an inflammatory-like response in gallbladder mucosa, as do proinflammatory agents such as lipopolysaccharide.68 The difference may be explained by the larger mass of cholesterol crystals embedded in the mucosa of patients with multiple CSs. Further investigations should aim at the identification of substances in cholesterol supersaturated bile that are likely to trigger increased expression of PLA2-II. In summary, this study suggests that altered physicochemical and biochemical composition, such as an increase in the LPC to PC ratio potentiating gallbladder mucosal inflammation, and increased total protein and hexosamine concentrations promoting cholesterol nucleation and increasing viscosity are more marked in gallbladder bile of patients with multiple CSs than in that of patients with solitary CSs. These alterations are associated with increased IR PLA2 -II levels in gallbladders and gallbladder bile and with increased gallbladder PGE2 concentrations.

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Received October 22, 1996. Accepted January 31, 1997. Address requests for reprints to: Junichi Shoda, M.D., Ph.D., Department of Gastroenterology, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba-shi, Ibaraki 305, Japan. Fax: (81) 298-53-3124. Supported in part by Grants-in-Aid for Intrahepatic Calculi from the Ministry of Health and Welfare, Japan, and Grants-in-Aid from University of Tsukuba Research Projects, Japan. Published in part in abstract form (Gastroenterology 1996; 110:A1325). The authors thank the following for their help and support throughout this work: Dr. K. Endow (Diagnostic Science Department, Shionogi & Co., Ltd., Osaka, Japan); Dr. T. Kato (Pharmaceuticals Research and Development Division, Shionogi & Co., Ltd., Osaka, Japan); Prof. N. Kobayashi (First Department of Surgery, Ehime University School of Medicine, Ehime, Japan); Prof. T. Fukao and his colleagues (Department of Gastrointestinal Surgery, Institute of Clinical Medicine, University of Tsukuba, Ibaraki, Japan); and K. Tomita (University of Tsukuba, Ibaraki, Japan) for her technical assistance.

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