effects of phospholipase a2, free fatty acids and 2-lysolecithin on the crystallization of...

European Journal of Clinical Investigation (2000) 30, 715±721 Paper 703

Effects of phospholipase A2, free fatty acids and2-lysolecithin on the crystallization of cholesterol ingallbladder bile

A. SchoÈne, D. JuÈngst, G. Meyer, T. Hernandez-Richter and S. Fischer

Klinikum Grosshadern, Ludwig±Maximilians-University, Munich, Germany

Abstract Background Phospholipase A2 (PLA2) and its enzymatic products free fatty acids (FFAs)

and 2-lysolecithin are physiological constituents of bile. Their role in the crystallization of

cholesterol in gallbladder bile of patients with cholesterol gallstones is still controversial.

Design To clarify this issue we evaluated the activity of PLA2 and the concentration and

pattern of FFAs in the gallbladder bile of cholesterol stone patients. We furthermore added

PLA2, FFAs and 2-lysolecithin to isotropic gallbladder bile, determined the crystal observa-

tion time (COT) and counted the cholesterol crystals in a crystal growth assay for up to 21

days.

Results A PLA2 activity of 1´8 6 1´2 U Lÿ1 and total FFA concentrations of 1´32 60´71 mmol Lÿ1 were determined. After incubation of bile for 24 h at 37 8C total FFAs

increased to 2´72 6 1´29 mmol Lÿ1 (P<0´005). Biliary saturated and unsaturated FFAs

were found in equal proportions before and after incubation, pointing to an additional

presence of lipases other than PLA2. A COTof 1 day was observed in all gallbladder biles and

increased to 1´7 6 0´5 days after addition of 5 U Lÿ1 of PLA2 (P<0´01). An even higher

COT of 2´5 6 0´8 days was seen after addition of 5 mmol Lÿ1 of a `biliary' mixture of

FFAs (P <0´005) but the COT remained unchanged after addition of 5 mmol Lÿ1 of 2-

lysolecithin. However, in the crystal growth assay in gallbladder bile addition of 5 U Lÿ1 of

PLA2, of 5 mmol Lÿ1 of `biliary' FFAs and of 5 mmol Lÿ1 of 2-lysolecithin decreased

signi®cantly the number of cholesterol crystals formed during follow-up.

Conclusion An elevated activity of PLA2 in gallbladder bile may counteract the formation

of cholesterol crystals through increased formation of FFAs and 2-lysolecithin. However,

regarding the comparatively low activity of PLA2 in gallbladder bile PLA2 seems to be of only

minor pathophysiological importance in the formation of cholesterol gallstones.

Keywords Crystal growth assay, crystal observation time, gas liquid chromatography,

phosphatidylcholines.

Eur J Clin Invest 2000; 30 (8): 715±721

Introduction

In cholesterol gallstone disease hypersecretion of biliary

cholesterol is likely to be the primary and major defect from

which other defects emanate [1]. These include gallbladder

hypomotility due to chronic in¯ammation and associated

release of mucin and proteins by the gallbladder epithelium

[2,3]. Phospholipase A2 (PLA2) is the most abundant

phospholipase and is present in the gallbladder wall and in

bile [4±7]. In¯ammation of the gallbladder wall might

induce an increase of PLA2-activity with subsequent release

of free fatty acids (FFAs) and lysolecithin from phospho-

lipids in gallbladder bile. Indeed, FFAs and lysolecithin have

been shown to be physiological components of gallbladder

bile and they do occur in the low millimolar range [8±13].

Previous studies on the in¯uence of PLA2, of FFAs and

of lysolecithin on the crystallization of cholesterol in bile

have been performed in model biles. No cholesterol pre-

cipitated when the lecithin in a bile salt solution saturated

with cholesterol was converted to lysolecithin and FFAs.

Q 2000 Blackwell Science Ltd

Department of Medicine II (A. SchoÈne, D. JuÈngst, S. Fischer);

Department of Surgery (G. Meyer, T. Hernandez-Richter),

Klinikum Grosshadern, Marchioninistrasse 15, D-81366

MuÈnchen, Germany.

Correspondence to: Sven Fischer, PhD, Department of

Medicine II, Klinikum Grosshadern, Marchioninistrasse 15,

D-81366 MuÈnchen, Germany. Tel.: �4989±70953130;

fax: �4989±7004418

Received 12 December 1999; accepted 22 April 2000.

716 A. SchoÈne et al.

This result suggests a minor effect of PLA2 on the crystal-

lization of cholesterol in bile. However, the model bile used

was not supersaturated with cholesterol [14]. Further

studies with PLA2 and with palmitic, oleic and linoleic

acids in model biles showed either prolonged or shortened

crystal observation times (COTs) [5,12].These con¯icting

results might be partially due to the use of model biles

instead of native gallbladder biles. Therefore, the in¯uence

of PLA2, of FFAs and of lysolecithin on the crystallization

of cholesterol in bile is still a controversial subject.

To investigate this issue further, we ®rst analysed the

activity of endogenous PLA2 in native gallbladder bile of

cholesterol stone carriers using radioactively labelled

lecithin as substrate. We then evaluated levels and pattern

of FFAs in native gallbladder bile before and after incuba-

tion to estimate endogenous lipase activities other than

PLA2. Furthermore, we monitored the effects of increasing

amounts of PLA2, of FFAs and of 2-lysolecithin added to

gallbladder bile on the crystallization of cholesterol.

Besides the COT we investigated the numbers of choles-

terol crystals formed during incubation of gallbladder bile

with PLA2, FFAs, or 2-lysolecithin in comparison to

negative controls. Thus, the principal aim of the study

was to evaluate the effects of PLA2, of FFAs and of 2-

lysolecithin on the crystallization of cholesterol in bile

under in vitro conditions as close to physiological as possible.

Materials and methods

FFAs, fatty acid methylesters, 2-lysolecithin and PLA2

were purchased from Sigma (Munich, Germany). Lecithin

labelled in the 2-position with [14C]arachidonic acid (speci®c

activity55 mCi mmolÿ1)was fromNEN(Dreieich,Germany)

and 3a-hydroxysteroid dehydrogenase was obtained

from Worthington Biochemicals (Freehold, NJ, USA).

All solvents were of analytical grade (Merck, Darmstadt,

Germany). The gas chromatograph was a 5160 Mega

Series from Carlo Erba (Milan, Italy). For analysis of

fatty acid methylesters a CP SIL 19 fused silica capillary

column (25 m, 0´32 mm I.D.) was used (Chrompack,

Middelburg, the Netherlands). Silicagel plates (0´25 mm)

for thin layer chromatography (TLC) were supplied from

Merck (Darmstadt, Germany).

Patients and collection of bile

Twelve patients with symptomatic cholesterol (>50%

cholesterol) gallstones were included in the study (three

males, nine females, age 28±68 years, single and multiple

cholesterol stones).

All patients gave informed consent after a detailed

explanation of the procedure required for intraoperative

bile collection. The patients did not take any drugs which

are known to be secreted into bile and might in¯uence

lithogenic factors in gallbladder bile. During laparoscopic

cholecystectomy the gallbladder was punctured and bile

was aspirated as completely as possible because of the

known strati®cation of human gallbladder bile [15]. All

native gallbladder biles showed microscopically visible

cholesterol monohydrate crystals. The absence of bacteria

and of blood in gallbladder bile was shown microbiologically

by bile culture and with the Haemoccult-test (Beckmann±

Coulter, Krefeld, Germany), respectively.

Microscopy and ultracentrifugation of

gallbladder bile

After the collection, bile samples were mixed thoroughly

and 5 mL was immediately examined by polarized light

microscopy for cholesterol crystals. Four millilitres of

gallbladder bile were centrifuged in polycarbonate tubes

with a 50 Ti rotor at 37 8C for 1 h at 100 000 g in a

Beckman L 5-65 ultracentrifuge (Beckman instruments,

Fullerton, CA) to precipitate biliary `sludge'. The super-

natant was microscopically free of cholesterol crystals.

Aliquots were incubated at 37 8C after addition of a trace

of an antioxidant (butylated hydroxytoluene, BHT) and

were examined daily with polarized light microscopy for up

to 21 days [16]. The time taken for cholesterol microcrys-

tals (plates) to appear was de®ned as the crystal observation

time (COT) and was measured in days.

Analysis of biliary lipids

Cholesterol was determined colorimetrically with the

Liebermann±Burchard reaction after double extraction of

1 mL of a methanolic bile sample with petroleum ether [17].

Phospholipids were measured as total biliary phosphate

after hydrolysis at 150 8C with sulphuric acid, using the

colorimetric assay of Fiske±Subbarow, and total bile salts

were determined by a modi®ed 3a-hydroxysteroid dehy-

drogenase method [18, 19]. The cholesterol saturation

index (CSI) of each sample was calculated in native bile by

dividing the cholesterol concentration by the maximum

cholesterol solubility according to Carey and was corrected

for the total lipid content of each individual bile [20].

For the determination of FFAs a trace of BHT as an

antioxidant and 5 mg of heptadecanoic acid (C17 : 0) as an

internal standard were added to 100 mL of gallbladder bile.

Then biliary lipids were extracted with chloroform/methanol

according to Bligh & Dyer [21] and the organic phase was

evaporated in a stream of nitrogen. FFAs were methylated

with diazomethane and fatty acid methylesters were separ-

ated by silicagel TLC from the other biliary lipids (solvent

system petrolether 40/60, acetone, glacial acetic acid,

95 : 4 : 1). Fatty acid methylesters were visualized by iodine

vapour, the silicagel was scratched off the plate and the

fatty acid methylesters were extracted with chloroform :

methanol : water [10 : 10 : 1]:. After evaporation of the

solvents, fatty acid methylesters were dissolved in ethyla-

cetate and analysed by capillary gas chromatography with

hydrogen as carrier gas. Biliary free fatty acids were calculated

according to the internal standard heptadecanoic acid

Q 2000 Blackwell Science Ltd, European Journal of Clinical Investigation, 30, 715±721

Phospholipase A2 and biliary cholesterol crystallization 717


(C17 : 0). The same method has been used previously for the

analysis of FFAs in hepatic bile using aliquots of 0´5 mL bile

[22].

Analysis of endogenous PLA2 in gallbladder bile

Activity of endogenous PLA2 in bile was calculated from

the appearance of free [14C]arachidonic acid from labelled

lecithin added to the gallbladder bile incubated at 37 8C for

24 h according to the formula described by Nakano et al. [4].

Formation of cholesterol crystals in isotropic

bile after addition of PLA2, of `biliary' FFAs and

of 2-lysolecithin

To 0´5 mL of isotropic bile traces of the antioxidant BHT

were added. Appearance and numbers of cholesterol

microcrystals were examined after 1, 2, 3, 4, 5, 6, 7, 14

and 21 days in isotropic bile on incubation at 37 8C and in

isotropic bile after addition of 5, 10, or 20 U Lÿ1 PLA2; of

5, 10, or 20 mmol Lÿ1 of a `biliary' mixture of FFAs (46%

C16 : 0, 37% C18 : 2, 4% C18 : 1, 8% C18 : 0 and 5%

C20 : 4) or of 5, 10, or 20 mmol Lÿ1 of 2-lysolecithin. Besides

the COT also the number of cholesterol crystals was exam-

ined microscopically with an 80-fold ampli®cation during the

time±course of 21 days. Cholesterol crystals were quantita-

tively observed in their time±course of formation.

Statistical analysis

Student's paired and unpaired t-test was used for statistical

evaluation. A P<0´05 was considered statistically signi®cant.

Data are given as xÅ 6 SD (table) or xÅ 6 SEM (®gures).

Results

Biliary lipids, CSI and COT in cholesterol

stone patients

Biliary cholesterol (16´1 6 4´2 mmol Lÿ1), phospholipids

(47´9 6 20´9 mmol Lÿ1), bile salts (107´5 6 58´1 mmol Lÿ1)

and total lipids (9´62 6 3´40 g dLÿ1) demonstrated well-

concentrated gallbladder biles of the 12 patients with

cholesterol stones. All native biles showed cholesterol-

monohydrate microcrystals, were viscous, brown±yellow

in colour and were free of bacteria and of blood. With a CSI

of 1´27 6 0´36 most gallbladder biles were supersaturated

and all developed newly formed cholesterol crystals within

1 day after ultracentrifugation.

Activity of endogenous PLA2 in native

gallbladder bile

Free [14C]arachidonic acid accounted for 20´5 6 8´8% of

total radioactivity after incubation of lecithin labelled in the

2-position with [14C]arachidonic acid with native bile for

24 h at 37 8C. In a control incubation without bile only

0´5% of radioactivity accounted for free arachidonic acid.

An activity of 1´8 6 1´2 U Lÿ1 was calculated for endogen-

ous PLA2 in gallbladder biles and PLA2 activity ranged

from 0´4 to 4´4 U Lÿ1.

FFAs in native gallbladder bile and after incubation

for 24 h

Concentrations and ranges of total FFAs and of the indi-

vidual FFAs in native gallbladder bile before and after

incubation at 37 8C for 24 h are shown in Table 1. Con-

centrations of FFAs were elevated two-fold after incuba-

tion. However, the pattern of saturated and unsaturated

FFAs after incubation of native bile remained unchanged.

FFAs in native gallbladder bile after addition of 5,

10, or 20 U Lÿ1 PLA2 and incubation for 24 h

Levels of total FFAs increased from 1´32 6 0´71 in native

bile to 9´78 6 4´58 mmol Lÿ1 after 24 h incubation with

5 U Lÿ1 PLA2. Linoleic and arachidonic acids contributed

nearly 75% of total FFAs. Concentrations of total FFAs

after incubation (24 h) with 10 and 20 U Lÿ1 PLA2 increased

to 11´33 6 3´14 and 12´70 6 2´9 mmol Lÿ1, respectively.

COTs in isotropic gallbladder bile after addition

of PLA2, of a `biliary' mixture of FFAs or of

2-lysolecithin

Cholesterol crystals developed in isotropic gallbladder

Table 1 Free fatty acids (FFAs) in native gallbladder bile of 12

patients with cholesterol stones before and after incubation for

24 h at 37 8C

Before After

Total FFAs (mmol Lÿ1) 1´32 6 0´71 2´72 6 1´29*

(0´53±2´53) (1´52±5´75)

Palmitoleic acid 0´03 6 0´03 0´08 6 0´06*

C16 : 1 2% 3%

Palmitic acid 0´60 6 0´32 1´19 6 0´47*

C16 : 0 45% 44%

Linoleic acid 0´37 6 0´23 0´72 6 0´47*

C18 : 2 28% 26%

Oleic acid 0´12 6 0´07 0´24 6 0´15*

C18 : 1 9% 9%

Stearic acid 0´09 6 0´03 0´18 6 0´06*

C18 : 0 7% 7%

Arachidonic acid 0´06 6 0´05 0´16 6 0´13*

C20 : 4 5% 6%

Eicosapentaenoic acid 0´02 6 0´01 0´04 6 0´03*

C20 : 5 2% 2%

Docosahexaenoic acid 0´03 6 0´02 0´08 6 0´04*

C22 : 6 2% 2%

*P<0´05.


biles within 1 day in all samples from cholesterol stone

patients.

After the addition of 5, 10 and 20 U Lÿ1 of PLA2 the

COTs were signi®cantly prolonged to 1´7 6 0´5, 1´7 6 0´5

and 2´0 6 0´6 days, respectively (Fig. 1a).

After the addition of 5, 10 and 20 mmol Lÿ1 of a `biliary'

mixture of FFAs the COTs were prolonged to 2´5 6 0´8,

3´2 6 0´9 and 4´5 6 1´8 days, respectively (Fig. 1b).

After the addition of 5, 10 and 20 mmol Lÿ1 of 2-

lysolecithin, however, the COTs remained unchanged

(Fig. 1c).

Numbers of cholesterol crystals in gallbladder bile

after addition of PLA2, FFAs, or 2-lysolecithin

Crystal growth curves of cholesterol crystals in gallbladder

bile after addition of 5 U Lÿ1 of PLA2, of 5 mmol Lÿ1

of a `biliary' mixture of FFAs, or of 5 mmol Lÿ1 of

2-lysolecithin in comparison to negative controls are

shown in Fig. 2(a±c). After addition of PLA2 the number

of crystals is signi®cantly decreased and the number of

crystals is lowest after addition of a `biliary' mixture of

FFAs. The addition of 2-lysolecithin signi®cantly

decreased the number of cholesterol crystals although the

COT did not change. Addition of 10 and 20 U Lÿ1 of PLA2

and of 10 and 20 mmol Lÿ1 of a `biliary' mixture of FFAs or

of 2-lysolecithin decreased the number of cholesterol crys-

tals even more (data not shown).

Discussion

All gallbladder biles in this study were obtained from

cholesterol stone patients undergoing laparoscopic chole-

cystectomy. They showed microscopically visible choles-

terol crystals in their native state and the ultracentrifuged

isotropic biles all had a COT of 1 day. Biles were well


Figure 2 (a) Formation of total cholesterol crystals in isotropic

gallbladder bile (n�6) without (W) and after addition (X) of

5 U Lÿ1 of phospholipase A2 (PLA2); *P< 0´05. (b) Without (W)

and after addition (X) of 5 mmol Lÿ1 of a `biliary' mixture of free

fatty acids (FFAs); *P<0´05. (c) Without (W) and after addition

(X) of 5 mmol Lÿ1 of 2-lysolecithin; *P<0´05.

Figure 1 (a) Crystal observation time (COT) in isotropic gall-

bladder bile (n�6) without and after addition of 5, 10, or

20 U Lÿ1 phospholipase A2 (PLA2); *P<0´01, **P<0´005. (b)

Without and after addition of 5, 10, or 20 mmol Lÿ1 of a `biliary'

mixture of free fatty acids (FFA); **P<0´005. (c) Without and

after addition of 5, 10, or 20 mmol Lÿ1 of 2-lysolecithin.



concentrated in their lipid content and were free of bacteria

and blood. Bacterial infection has been shown to enhance

levels of PLA2 and of FFAs in bile [4] and contamination

with blood may also alter biliary amounts of FFAs.

We found concentrations of FFAs in native gallbladder

biles in the range of 0´5±2´5 mmol Lÿ1 whereby palmitic,

linoleic, oleic, stearic and arachidonic acids were the pre-

dominant fatty acids. Concentrations of FFAs in hepatic

bile are lower by a factor of 50 as compared to gallbladder

bile and furthermore the pattern of the fatty acids is

different [22]. These ®ndings suggest that FFAs in gall-

bladder bile are not merely concentrated from hepatic bile

but are largely generated by the enzymatic action of phos-

pholipases on biliary lecithins. Lecithins are the main

phospholipids of bile and contain palmitic acid in the 1-

position and linoleic, oleic, or arachidonic acid in the 2-

position in nearly 75% of their molecular species [23]. In

this study the biliary levels of FFAs had doubled after 1 day

of incubation at 37 8C, but the pattern of FFAs with nearly

equal amounts of saturated and unsaturated fatty acids had

remained the same, indicating that different phospholi-

pases are active in native bile. Besides the main lipolytic

enzyme PLA2, phospholipase C, lysophospholipase (which

is able to hydrolyse 1-acylphosphoglyceride) and dia-

cylglycerollipase activities have also been described in bile

[5,24±26]. Furthermore, when the gallbladder no longer

has an adequate function and there is stasis of bile the levels

of FFAs and of lysolecithin may even increase more by the

action of phospholipases embedded in the gallbladder

epithelium [24]. Whereas the levels of FFAs in native or

incubated bile are generated by the mixed activities of

different lipases and phospholipases, we were able to

calculate exactly the activity of PLA2, presumably the

most active phospholipase in bile, by using lecithin radio-

labelled in the 2-position with arachidonic acid. The

medium activity of 1´8 6 1´2 U Lÿ1 was in the range of

that found previously in the bile of cholesterol stone

patients and was larger than the activity of phospholipase

C reported previously for bile by a factor of 10 [5]. The

existence of biliary saturated FFAs in equal amounts as

unsaturated fatty acids points to additional activities of

phospholipase A1, of lysophospholipase or of diacylglycerol-

lipase in bile which can all cleave saturated fatty acids from

the 1-position of the glycerol skeleton [26].

For our studies on the crystallization of cholesterol in

bile we chose PLA2 as it is the most abundant phospholi-

pase in bile and is more active than phospholipase C or D

by a factor of 10 [5±6]. Upon adding supraphysiological

activities of PLA2 to isotropic bile the rapid COTs of

cholesterol were signi®cantly prolonged. We found that

the concentrations of FFAs did not raise in parallel but

reached an almost constant level, probably due to product

inhibition of the enzyme. Thus increasing levels of PLA2

have a signi®cant antinucleating effect on cholesterol in

gallbladder bile as documented by a prolongation of the

COT. When only a physiological mixture of `biliary' FFAs

was given in increasing amounts to isotropic bile the COTs

of biliary cholesterol rose even more, to a mean of 4´5 days.

In contrast, increasing amounts of 2-lysolecithin had no

effect at all on the COT of cholesterol in bile. Thus the

generation of FFAs and of 2-lysolecithin by increasing

levels of PLA2 has a less pronounced prolonging effect on

the COT of cholesterol than the addition of `biliary' FFAs

alone. The effects of FFAs on the COT might be mediated

by their ability to further solubilize micellar and, more

importantly, vesicular cholesterol. However, to our knowl-

edge the solubility of cholesterol in the quaternary system

of cholesterol, phospholipids, bile salts and FFAs with

concentrations of FFAs in the physiological range of

about 1 mmol Lÿ1 has not yet been determined. In con-

trast, 2-lysolecithin does not possess such potent solubiliz-

ing properties. In experiments with model bile a lower

solubilizing potency of 2-lysolecithin as compared to

lecithin for cholesterol was found. No cholesterol precipi-

tation, however, was observed when lecithin was converted

to 2-lysolecithin in a bile salt solution [14] supporting our

results upon addition of 2-lysolecithin to isotropic bile.

The COT is a key factor in the pathogenesis of choles-

terol gallstone disease and predicts the propensity of iso-

tropic gallbladder bile to form cholesterol crystals [16].

However, it is not a quantitative criterion and does not tell

us about the amount of crystals formed in isotropic bile

during a period of time. A spectrophotometric method

measuring the optical density of a cholesterol crystal dis-

persion has been introduced as a crystal growth assay in

model biles mainly to test protein promotors of crystal-

lization [27,28]. However, this crystal growth assay depend-

ing on an optical method is not suitable for native

gallbladder bile because of its interference with the pig-

ments of natural bile. Our method of counting the crystals

in 5 mL of bile at an 80-fold magni®cation yields a result of

the crystal numbers formed in the course of the days.

Although the method is cumbersome one can clearly

demonstrate an inhibitory effect of PLA2, FFAs and 2-

lysolecithin on the number of cholesterol crystals formed

during the observation period in pigmented gallbladder

bile. Whereas in isotropic bile after 7 days a maximum of

approximately 800 crystals mlÿ1 had formed, the numbers

of crystals after addition of physiological amounts of PLA2,

FFAs and 2-lysolecithin was below 200. After 1 week of

observation the numbers of crystals in bile without any

additives decreased because the isolated cholesterol crys-

tals formed clusters and aggregates. In our opinion the

number of cholesterol crystals or the mass of cholesterol

crystals precipitated from bile after 1 week is a quantitative

factor indicating the lithogenicity of bile more precisely

than the qualitative factor of COT. Furthermore, by count-

ing the numbers of cholesterol crystals after the addition of

other effective substances the pro- and antinucleating

properties of these substances can be evaluated by this

novel and physiological crystal growth assay in gallbladder

bile.

As PLA2 is the phospholipase most active in gallbladder

bile [5] our in vitro experiments should be representative

for the complex in vivo situation of cholesterol crystal-

lization in gallbladder bile. According to our experiments

PLA2 prolongs the COT of cholesterol in gallbladder

bile by its enzymatic products FFAs and 2-lysolecithin.


Furthermore, the total number of cholesterol crystals

formed in 1 week in gallbladder bile is strongly reduced

in the presence of PLA2 and its enzymatic products FFAs

and 2-lysolecithin. In our experimental design supraphys-

iological amounts of PLA2, FFAs and 2-lysolecithin were

used to cover also the pathophysiological situation of

gallbladder in¯ammation. In this range of PLA2 activity

in bile the induced release of FFAs and 2-lysolecithin from

biliary phospholipids has a distinct antinucleating effect on

the crystallization of cholesterol in gallbladder bile. Regard-

ing our results presented in Fig. 1 it is reasonable to suggest

that the addition of lower physiological amounts of PLA2

activity would have a less pronounced antinucleating or

even no effect on the formation of cholesterol crystals in

gallbladder bile. Thus, increased activities of PLA2 in

gallbladder bile in patients with acute or chronic in¯am-

mation of the gallbladder wall do not favour directly the

formation of cholesterol crystals and cholesterol gallstones.

These ®ndings in in vitro studies using native bile do not

exclude indirect effects via stimulation of mucin secretion

of the gallbladder epithelium by FFAs or the well-known

role of an increased PLA2 activity for the formation of

brown pigment gallstones [29]. Accordingly, an increased

activity of PLA2 in gallbladder bile counteracts the forma-

tion of cholesterol crystals via increased amounts of FFAs

and 2-lysolecithin. Regarding, however, the low activity of

PLA2 in gallbladder bile PLA2 seems to be of only minor

pathophysiological importance in the formation of choles-

terol gallstones.

Acknowledgements

The authors wish to thank Benedikta ZuÈndt and Gudrun

Schwertfeger for their excellent technical assistance. Part of

this work was presented at the 47th and 48th Annual

Meetings of the American Gastroenterological Association,

Washington D.C. (May 1997) and New Orleans (May

1998) (Gastroenterology 1997;112:A505 and Gastroenterol-

ogy 1998;114:A540). This work was supported by the

Friedrich±Baur-Foundation, Munich, Germany.

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effects of phospholipase a2, free fatty acids and 2-lysolecithin on the crystallization of...

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