Stability of human gallbladder bile : Effect of freezing

1Department of Internal Medicine, Krankenhaus Burg, Burg; 2University of Ulm, Department of Internal Medicine I, Ulm, Germany Correspondence and reprints: Dr Paul Janowitz, Krankenhaus Burg, Academic Teaching Hospital of the University of Magdeburg, August-Bebel-Straße 55a, 39288 Burg, Germany. Telephone +00493921-96-1301, fax +00493921-96-1340, e-mail Janowitz@freenet.de Received for publication September 26, 2000. Accepted January 22, 2001 P Janowitz, R Mason, W Kratzer. Stability of human gallbladder bile: Effect of freezing. Can J Gastroenterol 2001;15(6):363366. In the present study, the stability of the most essential biliary parameters of human gallbladder bile at 18°C was examined over several months. In 12 patients with gallstone disease (10 female, two male; 52.1±13.3 years of age), bile was obtained through fine needle puncture of the gallbladder under local anesthetic. The concentrations of total lipids, cholesterol, phospholipids and bile acids, and the cholesterol saturation index and crystal appearance time were determined before and after freezing over a mean period of 4.38±2.9 months. Gallbladder bile obtained by fine needle puncture has proved to be of excellent quality. The total lipid concentration was unchanged before (8.30±4.16 g/dL) and after freezing (9.16±4.54 g/dL, P=0.6027). The biliary cholesterol, phospholipids and bile acid concentrations, and cholesterol saturation index showed no statistically significant differences before and after freezing. A significant difference arises in the context of subdivision of the group to the nucleation time. Before freezing, most patients had a nucleation time between five and eight days, which shortened to between one and four days after thawing (P=0.0100). The authors conclude that, with the exception of the nucleation time, human gallbladder bile can be stored at 18°C for four months with stability of major lipid components.

T he ongoing progress in the conservative therapy of gall- stone disease has focused attention on the importance of adequate patient selection (1)(2)(3)(4)(5)(6).Human gallbladder bile samples are often used in biliary research.The presence of cholesterol crystals and the determination of nucleation time (NT) in the gallbladder bile are the best methods of distinguishing between cholesterol and pigment gallstones, and correlate well with computed tomographic analysis (1,7,8).Furthermore, microscopic examination of bile is easier, quicker and cheaper than biochemical analysis of bile or computed tomography of the gallbladder (9)(10)(11)(12).An immediate examination of the gallbladder bile at 37°C is considered a prerequisite for a microscopic examination.Conjugated bilirubin remains relatively stable at -70°C over several months but degrades rather quickly at -15°C (13).Other biliary parameters, such as bile acids, phospholipids and cholesterol, are stable at -30°C over a week (14).The aim of the present study was to examine the stability of the major lipid components at -18°C over several months.

PATIENTS AND METHODS
In 12 patients with gallstone disease (10 female, two male; 52.1±13.3 years of age; five cholesterol and seven mixed pigment gallstones), bile was obtained through fine needle puncture of the gallbladder under local anesthetic.In one female patient, the gallbladder puncture was performed twice.
Bile aspiration was performed for research purposes; therefore, appropriate informed consent was obtained from each patient.Patients with a history of chronic or acute cholecystitis, or sonographically or computed tomographically confirmed cholecystitis were excluded from the study.
The fine needle puncture of the gallbladder was performed between 08:00 and 09:00 after a 12 h fasting period.The method has been described elsewhere (1,3,15).The transducer was equipped with a commercially available lateral puncture attachment to provide a biopsy guide for all punctures.To avoid bile leakage, the gallbladder bile was completely aspirated in each patient.Aerobic and anaerobic cultures were performed immediately after aspiration.The positive samples contaminated by bacteria have been excluded from the present study.The bile samples were ultracentrifuged at 100,000 g (37°C) for 2 h.The isotropic (cholesterol-and liquid crystal-free) interphase was incubated at 37°C afterwards.The NT was determined by applying the method of Holan et al (8), defined as the earliest time required for detection of cholesterol monohydrate crystals in aliquots of the incubated isotropic phase, expressed in days.One drop of incubated aliquots was examined daily on a glass slide at a magnification of 100× to 400× under crossed polars (Leitz, Germany).Total biliary bile acids were determined by enzymatic testing (Sterognost-3-alpha, Nycomed N-0401 Oslo 4, Norway).Bile acid analysis was carried out using high performance liquid chromatography with dexamethasone as the internal standard (16).The biliary cholesterol and phospholipid concentrations were determined using an enzymatic colour test (CHOD-PAP method, Boehringer Mannheim, Germany).The total lipid concentration of bile (TLCO) is the sum of the cholesterol, bile acid and phospholipid concentrations, and is expressed in g/dL (conversion factors: cholesterol 386 g/mol, bile acids 495 g/mol, phospholipids 735 g/mol).The cholesterol saturation index, as defined by Admirand and Small (17), was calculated using a microcomputer program described by Kuroki et al (18).The rest of each gallbladder bile sample was stored at -18°C over a mean period of 4.38±2.9months (range 0.3 to 13 months).After thawing at room temperature over 12 h, the bile samples were ultracentrifuged at 100,000 g (37°C) for 2 h; the isotropic interphase was then incubated at 37°C, and the renewed determination of the biliary bile parameters was carried out according to the methods described above.
All data are presented as mean ± SD.Statistical significance was calculated using the Student's t test for dependent random samples.

RESULTS
The gallbladder puncture was successful in all 12 patients (100%).Complications due to the fine needle puncture, such as bleeding, bile leak or inflammation, were not observed.The TLCO was unchanged before (8.30±4.16g/dL; range 1.38 to 15.3 g/dL) and after freezing (9.16±4.54d/dL; range 1.41 to 16.4 g/dL) (P=0.6027).There was no significant correlation between lipid concentration and the length of the storage period (r=-0.0704,P=0.8194).Other biliary components also showed no statistically significant differences before and after freezing (Tables 1 and 2).
The NT was not significantly different in the complete group before freezing with 7.9±7.5 days and after freezing with 5.5±7.9days (P=0.4236);however, individual fluctuations have been observed (Figure 1).A significant difference arises in the context of subdivision of the group for the NT (P=0.0100,χ 2 test).Before freezing, most patients had an NT of between five and eight days.After thawing, this peak lay between one and four days.

DISCUSSION
In the present study, the stability of human gallbladder bile remained unaltered at -18°C over a mean period of 4.38±2.9months.Cholesterol, phospholipids, bile acids, total lipid concentration and the cholesterol saturation index showed no trends or significant changes.Other studies had already shown that biliary components are stable at -30°C over one week (14).In a study of porcine gallbladder bile, the pH and concentrations of total and ionized calcium, phospholipid, cholesterol and bile salts were stable over 17 days at -17°C (19).The total bilirubin concentration was also stable at -15°C (19).
The NT was the only parameter that showed clear individual fluctuations.Before freezing, most patients had an NT between five and eight days; after freezing this peak was between one and four days.In the initial stage of cholesterol gallstone formation, the nucleation of cholesterol monohydrate crystals occurs within the mucous gel (20).The results of other studies suggest that qualitative differences in individual proteins of gallbladder bile are responsible for nucleation-promoting activity in vitro (20)(21)(22)(23)(24). On the other hand, the purified apolipoproteins A-1 and A-2 are able to prolong the NT (25)(26)(27).Apolipoprotein A-1 stabilizes the nonmicellar fraction by forming an apolipoprotein A-1 and lipid complexed particle, resulting in prolonged cholesterol crystal nucleation (27).The close association of vesicles and crystals in human gallbladder bile supports the contention that vesicles are important in the initial nucleation of cholesterol monohydrate crystals (28).Either the glycoprotein activity becomes strengthened after freezing or the activity of the nucleation inhibitor factors is reduced.In the present study, the glyco-proteins and the apolipoproteins were not measured; therefore, it is not possible to comment on this.The prerequisite for a longer storage of the gallbladder bile is presumably an extraction of the samples without bacterial contamination.In another study, the gallbladder bile was contaminated in 13.3% of patients with gallstones without history and without evidence of acute or chronic cholecystitis (29).

CONCLUSION
With the exception of the NT, human gallbladder bile can be stored at -18°C for four months with stability of major lipid components.Submit your manuscripts at http://www.hindawi.com

Figure 1 )
Figure 1) Distribution of the nucleation time in days before and after freezing (n=13)