57 I Aurf. N.Z. J . Sarg. 1989.59. 571-577

---- SURGICAL RESEARCH

BACTERIA AND GALLSTONE NUCLEATION LUIS vITElTA, '* A V N l SALI,** VICKY M O R l n , 3 t ANDREW SHAW,4t

PETER CARSON,'+ PETER LllTLE6* A N D AYMAN E U A R K A 7 * * Universiry of Melbourne Department of Surgery, and t Microbiology Unif P~rh01og.v Dcpurtmettr.

Repatriation General Hospital. Heidelberg, Victoria This preliminary study reports for the first time that there might be a possible association between bncteria and the aetiology of some cholesterol calculi. The gall-bladder biles from 225 cholecystectomy patients underwent bacteriological and microscopic study. Cholesterol calculi from 13 patients (10.2%) were observed to be associated with gall-bladder bile profusely infected with at least one bacterial species that was shown to possess /3-glucuronidase activity, an enzyme that i s thought to promote calcium bilirubinate precipitation in bile. Concomitantly, the associated gall-bladder bile was 'high' in calcium bilirubinate precipitation and the precipitate was also detected in the centre of the gallstones. Moreover, in approximately half of these patients (six of I3), the cholesterol gallstones' nuclear areas also contained calcium palmitate. which i s also thought to be due to bacterial activity.

The results also support the hypothesis that bacteria with active /3-glucuronidase (for example, Esdterirhicr coli) can significantly influence the aetiology of brown pigment gallstones. In contrast, bacteria were observed to have no role in black pigment gallstone formation, as their associated gall-bladder biles were always observed to be sterile.

Key words: aetiology, bacteria, &glucuronidase, gallstones.

Introduction On the basis of morphology and chemical composi- tion, gallstones have been classified into one of two major types, namely, cholesterol or pigment cal- culi. Cholesterol calculi occur as either 'solitaire' or multiple forms.' Pigment calculi can also be divided into two groups. namely, brown and black pigment.

Gallstones occur in a variety of shapes and sizes with crystalline to amorphous structures, and gen- erally are perceived to grow gradually by sedi- mentation around an initial nucleus. Pigments like calcium bilirubinate (CaB) as well as mucus lyco- proteins have been found in gallstone cores? Cal- cium and bilirubin can associate with mucus and it is thought that these complexes can in turn act as initiating factors for heterogeneous cholesterol nu- cleation o f supersaturated bile. '-'

I t has been suggested that bacteria play a crucial role in the aetiology of brown pigment gallstones in

'F'hD: Surgical Rewarch Fellow. 'MB. BS. PhD. FRACS. FACS; First Assistant Surgery. .'MSc. MASM; Microbiologisr. ' RSc(Huns): Microbiulogist. ' AIMLS. ARACI; Senior Microbiologist. I, MB. BS. FRACS; Research Associate. ' MB. BS. Reearch Fellow.

Correspnndcncc: Dr L. Vitetta. University Department of Surgery. Rcpatriatiun General Hospital. Heidelberg. Vic. 3081. Auslralia.

Accepted for publication 6 January 1989.

Oriental countrie~.~.' In Japan, where brown pig- ment gallstones are commonly found, CaB is the predominant component of these gallstones. In investigating these stones, Maki observed that the gall-bladder biles were invariably associated with Escherichia coli, and that the bile had active j3- glucuronidase activity, which was demonstrated to be of bacterial origin.7 Subsequent experiments in which E. coli was incubated with human bile showed that a sediment consisting chiefly o f CaB was produced. Maki thus linked E. coli active /I- glucuronidase, CaB precipitation in bile and brown pigment gallstones with the characteristically high content of CaB. He subsequently postulated that p- glucuronidase deconjugated bilirubin diglucuronide to free bilirubin in bile, which then precipitated as CaB, and acted as a nidus for brown pigment gall- stone fo~mation.~ Recently, Cetta suggested that, in addition, bacteria also elaborate via a phos- pholipase, calcium palmitate (CaPalm) which has been detected with CaB in recument brown pigment stones in the bile-duct o f patients from Western countries .9

Having a Western-type population and environ- ment, this study set out to examine the role o f bac- teria with active /%glucuronidase and its influence on gallstone formation. Gallstones from patients undergoing cholecystectomy were analysed, bile was examined microscopically, and these findings were correlated with the presence of bacteria and their j3-glucuronidase activity.

512 VITETTA €7 A L

Met hods

I ' A T l t S I S

The subjects studied were 225 patients admitted because of gallstone disease. to the Repatriation General Hospital Hciklberg. Melbourne. Australia (Table I ) . The patients were admitted to this hospi- tal during the period December 1981-October 1986 (although no patients admitted during 1984 were included). There were 97 males. mean age 58.5 years (s.d. = 12.2) and 128 females. mean age 53.3 years is.d. = 15.6). The majority (98.7%) were of European descent. with the re- mainder ( I .3'% ) being from South-East Asian countries.

Table 1. Frequency of positive bile cultures and type of gallstone case

Gallstones only Acute cholecystitis Gallstones and bile-duct

\tone\ with obstructive jaundice

Mucwrle of the gall-Madder

Acalculous cholecyst itts

NO Pomve culture ( "" )

171 28. I 43 27.9

_____ --

9" 88.9'

6 16.7' S 40.0'

'These patients were ;ilw includcd in ~hc gall\tonc\ onl)

' Thc nunibcr of pslieni\ wa* t w \niall fo ohtain an accuratc p u p .

ectimrie ol' percentage

I s . r o N E s

Gallstones were initially examined for their gross morphological characteristics and were subse- quently classified into one of four groups (Fig. I ) . Infrared spectroscopy was used to detect the com- mon gallstone coniponcnts. namely. cholesterol. bilirubin. and calcium. the latter in the form of the hilirubinate. carbonate. phosphate and palmirate salts (Tables 2. 3) .

Reference infrared bpectra of cholcstcrol. cal- cium carbonate (CaCarb) and calcium phosphatc (CdPhos). were recorded on high purity standards (Sigma). CaB and CaPalm were prepared according to previously described methods. l " - tz The infrared spectroscopy method was that adoptcd from Trot- man rf c t / . ' 7 An infrared spectrum was obtained on the gallstoneMRr wafer with a Perkin-Elmer 157 prating infrared qwtrophotometer.

H I I. t.

Following ligation and section of thc cystic duct at operation. gall-bladder bile was mixed thoroughly. and totally aspirated transmurally with a syringe

from the gall-bladder, and was subjected immedi- ately to both aerobic and anaerobic culture. and microscopy.

Fig. 1. Types of gallstones. (a) cholesterol single; (b) cholesterol multiple (from sterile bile, left. and from bile with positive culture for p-plucuronidase active bacteria); (c) brown pigment: (d) black pigment. Scale in cm.

Table 2. Cholesterol giallstones: IR spectroscopy and ~--plucurontdase active bacteria in bile

Gallstone types Cholesterol Cholesterol

sol i tary multiple 01 = 40) 0 1 = 128)

. __ . - . - .

/j-plucumnidase Absent Absent Present activc bacteria in 40 in 115 in I 3

IR profile* CaB nucleus only 2 31 13 CaCarh

nucleus 3 36 nd middle I 9 nd surface -I 24 nd

nucleus nd nd 6 middle nd nd nd wrl'ace nd nd nd

nucleus nd 2 nd middle nd nd nd wrl'ace nd nd nd

* Chcilcwrol was presenf in a l l wlitary and multiplc siinplc\

CiPiilm

CilPhos

:mJ wa\ cli\trihuted diffuwly: nd: n a detected.

BACTERIA AND GALLSTONE NUCLEATION s73

Table 3. Pigment gallstones: 1R spectroscopy and B- glucuronidase active bacteria in bile

Gallstone type Brown pigment Black pigment

(n =22) (n = 29)

p-glucuronidase Present Absent active bacteria in 22 in 29

IR profile Cholesterol

nucleus 7 middle I 2 8 surface 14

nucleus 22 middle 22 surface 22

nucleus nd middle nd surface nd

nucleus 22 middle 18 nd surface 18

nucleus nd middle nd 9 surface nd

CaB

CaCarb

CaPalm

CaPhos

14

17

nd: not detectcd.

BILE Clll.TllRE

Gall-bladder bile samples were examined for bacte- ria by Gram's stain. Bile was directly plated onto sheep blood agar (SBA), McConkey's agar, horse blood agar (HBA), lysed horse blood with Van- comycin and Vitamin K (LKV), Nagler's agar, phenyl-ethyl alcohol agar, and cooked meat medium. HBA, LKV, Nagler, phenyl-ethyl alcohol agar and cooked meat medium were incubated anaerobically in jars flushed with a commercial gas mixture (85%N2, 5YoHz, IOYOCO~). All plates were incubated at 35°C for 5 days and examined daily for growth.

BACTERIAL 6-GLUCURONIDASE ACTIVITY

To detect the activity of bacterial &&curonidase, all bacterial species isolated from the gall-bladder biles were subcultured and the enzyme was estimat- ed qualitatively by the previously described method of Tabata and Nakayama.8 Briefly, the hydrolytic activity of B-glucuronidase was determined by in- cubating the bacteria, previously grown in a nutri- ent broth, with the substrate. p-nitrophenyl-p-0- glucuronic acid, for 1-4h at 37°C. A positive test for j3-glucuronidase activity was indicated by the development of yellow colour.

B I L E MICROSCOPY

Bile samples from the gall-bladder were examined for biliary crystals (cholesterol (Chc)) and CaB within 5 min of Biles were mixed thoroughly and placed in test tubes in a water bath at 37°C. To demonstrate even mixing, a drop from the top. middle and bottom of the tube was exam- ined microscopically in direct and polarized light at low (x 100) and high (x 400) magnifications (Fig. 2).

Fig. 2. Microscopy of gall-bladder bile with positive cul- ture for E. coii from gall-bladder with multiple cholesterol gallstones. Magnification factor X 100 in direct light.

Gall-bladder biles were first examined micro- scopically to detect monohydrate Chc and subse- quently noted if the cholesterol precipitation was low, moderate, or high at low magnification ( x 100) under polarized light. The presence of CaB granules was confirmed by comparison with syn- thetic CaB samples synthesized in the laboratory by the methods of Edwards er ul. as well as Sutor and Wilkie, and chemically verified by infrared spec- troscopy. 1''*11 As previously described by Juniper and Burson, CaB pigment ranules occur in bile as

tation in bile was defined as CaB granules present in 3 75% of the microscopic field of view at low magnification (x 100). and low pigment precipita- tion in bile was defined as CaB granules present in S 25% of the microscopic field of view at low magnification (x 100).

Results

yellowish-brown clumps.'. F High pigment precipi-

GALLSTONES

Gallstones examined by infrared spectroscopy showed the presence of cholesterol, CaB, CaCarb.

574 VITETTA ETAL.

CaPalm and CaPhos. In most gallstones. it was possible to investigate separately the distribution o f components in the nuclear. middle and surface re- gions. Al l cholesterol gallstones contained. as expected, cholesterol that was distributed diffusely. A variety o f calcium salts were also detected in these calculi. o f which CaCarb and CaB were the predominant calcium precipitates. These calcium salts usually occurred in cholesterol gallstone nu- clear areas. I n six patients with multiple cholesterol gallstones. CaB in the gallstone nuclear areas was detected in conjunction with CaPalm (Table 2).

In brown pigment gallstones. cholesterol was also detected, occurring predominantly in the mid- dle and surface areas (Table 3). CaB was the most predominant calcium precipitate. and i t was detect- ed in every sample that was distributed diffusely. CaB in these calculi was in most cases (82%) cou- pled to CaPalm. throughout the gallstone. How- ever. in the nuclear areas. CaB was always detected with CaPalm (Table 3). The amorphous nature o f black pigment gallstones did not allow a detailed investigation o f thcir components' distribution. CaB. CaCarb and CaPhos were detected regularly (Table 3 ) .

II I I. b. cis i:r I'K ES

In 225 patients. positive bacterial cultures occurred in 31.1% of biles. Patients with brown pigment gallstones had an incidence o f positive bacterial culture in bile o f 100'%. Two-thirds o f these bile samples were associated with more than one bacte- rial strain. Every gall-bladder bile sample associated with the% brown pigment gallstones was infectcd with at least one bacterial spccics that was active for 9-plucuronidase. In patients with cholesterol gallstones. the incidence o f positive bacterial cul- tures in bile was much less. with rates of 10.0% and 33.h'% for the cholesterol solitaire and multiple cholesterol gallstone types. respectively. Slightly more than half (51%) of the positive bile cultures lrom the patients with multiple cholesterol gall- stones were associated with more than one bacterial strain. In 13 patients with niultiplr cholesterol gall- stone%. the gall-bladder biles were associated with at Icast one bacterial species that was active for /I- glucuronidaw. In contrast. the patients with black amorphous pigment gallstones had sterile gall- bladder bilcs (Tables 4. 5 ) .

A number of aerobic and,anaerobic bacterial spe- cies was isolatcd from thc positivc culturcs of 225 gall-bladdcr b i b . Thc niosr commonly occurring specie\ were E. 1 4 . Kleb.~rc~llir pticiitnoiiiiw. Smp- towccri.\ fcwt rrlis . and Closiri~iitotc ~~cr/i.ingc.c~ii.r (Table 6 ) .

Quantitative estimates * h o w 4 that all positive

Table 4. Characteristics of cholesterol gallstones

Cholesterol gallstone type Solitary Multiple ( n = 40) ( n = 128) -

Malesfernales 12:28 55:73 Age (years)

Male5 58.6 f 12.1 56.7 2 12.1 Females 52.6 f 13.7 52.3 2 15.8

Bactibilia 4 (10.0%) 43 (33.6%) 13-glucuronidase

active bacteria present 0 (0.0%) 13 (10.2"/0)

Sterile bile 36 8S

Chc 40' 128"' Bile microscopy

CdB precipitate 1kglucuronidase

active bacteria present 0 13'

fi-glucuronidase active bacteria absent I' I o*

hierile bile 8* 24 *

Dcgrce ul precipitation: * low. 'moderate (Chc only). 'high. Dicta on age arc cxprcwxl a> m a n and \ d

Table 5. Characteristics of pigment gallstones ~~ ~ ~

Pigment gallstone type Brown Black (n = 22) ( n = 29) -

Males:females Age (years)

Males Females

Bactihilia B-glucuronidase

active bacteria present

Sterile bile Bile microscopy

Chc CaR precipitate

13-glucuronidase active bacteria present

/j-glucuronidase active bacteria absent

sterile bile

14:8 1415

65.7f 11.2 511.32 12.1 58.4 2 20.4 57.5 f 14.4 22 (loo0/") 0 (O.O0/0)

0 (0.0%) 22 (loo'%>) 0 29

22* 2*

2 2 i -

Dcgrcc of precipitation. * low. 'high Data on age are cxprcswed a\ iiiean and r.d.

gall-bladder bile cultures were greater than loo0 colony forming units (CFU) per mL of bile, with n w t having b IOOOOCFUlmL of gall-bladder bile.

BACTERIA AND GALLSTONE NUCLEATION 57s

BACTEHIAI. fl-GLUCURONIDASE ACTIVITY

Bacterial P-glucuronidase was detected in a number of bacterial strains that included E. coli, K . oxy- ioca, Bacreroides fragilis, and C . perji-ingens (Table 6).

Table 6. Bacterial species isolated from the gall-bladder biles of patients with cholelithiasis

Bacterial type No.

Aerobic species Escherichia coli* Klebsiella K. pneumoniae K . oxyroca* K. ozaenae

Enrerobacter E . aerogenes E. cloacae E. gergovia E . agglomerons

Proreus mirabilis Streptococcus

S . liquefaciens S . bovis S . viridans S. faecalis S. mitis S. milleri S . sulivarius S. sanguis

Staphylococrus S. hominus S . aureus S . warneri S . epidermidis

Citrobacrer freundii hcrobacillus casei Serratia liquejuciens Aeromonas hydrophila Morganella morganii Pseudomonas aeruginosu Corynebacrerium spp. Candida albicans (aerobic yeast)

Bacreroides fragilis* Closrridium perfringens* Propionibucterium ucnes

Anaerobic species

27

12 4 I

I 5 I 2 7 2 I 3 I I 2 2

3 10

I -

* All with P-glucuronidase activity

BILE MICROSCOPY

When gall-bladder bile was observed under direct and polarized light at low power ( X IOO), 15.6% of the gall-bladder bile samples had high CaB pig- ment precipitation, and 84.4% of the bile samples had low CaB pigment precipitation or none. Gall- bladder biles with high CaB pigment precipitation were associated with all brown pigment gallstones, 10.2% of the multiple cholesterol gallstones, and

with the gall-bladder bile of one patient with acal- culous cholecystitis (from six such cases found). Gall-bladder biles with high CaB pigment precipi- tation were all positive cultures for /3-glucuronidase- possessing bacteria. Gall-bladder biles with low pigment precipitation were associated with CaB precipitates that were observed to occur in gall- bladder biles infected with bacterial strains shown not to possess the &glucuronidase capacity as well as in sterile gall-bladder biles. However. in these instances the precipitate was present in significantly reduced proportions.

Discussion Studies from Japan and Italy have suggested that bacteria play a pivotal role in the formation of CaB and CaPalm.”x*Y These calcium salts are important components of brown pigment gallstones which have been associated with bacterial overgrowth and activity.’

In the present study. therefore, initial investiga- tions consisted of morphological examinations of brown pigment gallstones from 22 patients (9.8% of the total). These calculi had a layered appearance and were pigmented. Infrared spectroscopy con- firmed that CaB was a major component, diffusely and uniformly distributed throughout the gallstone. CaB was always associated with CaPalm in the nuclear areas, whereas, in extranuclear areas, this was not always the case. Bacteriological results correlated with those o f previous studies that have shown that brown pigment gallstones are usually associated with a bactibilia of 100%.2 but. most importantly, i t was observed that at least one bactc- rial isolate which had active /3-glucuronidase activ- ity was detected in each bile sample. Thus, i t was established that brown pigment gallstones had abundant CaB deposits throughout their structure. and that their associated gall-bladder biles had bacterial overgrowth for at least one species with active ,&glucuronidase, with abundant CaB pre- cipitation. This result follows previous studies which strongly suggest that bacterial hydrolytic activity in bile influences the aetiology of brown pigment gallstones.

The results of this study demonstrate and confirm the work of previous studies that have shown that bacteria are not associated with black pigment gall- stone fo rma t i~n . ’~ - ’~ These results were a good bacteriological internal standard. as the bile associ- ated with these gallstones was always observed to be sterile. Although microscopic examination of bile showed the occasional small Chc and CaB clumps, bile associated with black pigment gall- stones mostly showed no significant amounts o f either crystal type. Thus, black pigment gallstones

576 VITETTA ET AL

were associated with sterile bile and. as a come- quence. bacteria have no role in their formation.

As this serics of Australian patients with cholelithiasis also comprised those with cholesterol gallstones. both CaB and CaPalin were also detec- ted by infrared spectroscopy in a numbcr o f multi- ple cholcsterol gallstones. This led to preliminary findings which suggested that bacteria with active 8-glucuronidasc could play a significant role in the formation of gallstones by a mechanism similar to that postulated for brown pigment gallstones.

I n six and seven paticnts. respectively. CaB associated with CaPalin and CaR alone. were ob- scrvcd in the nuclear areits of their cholesterol gall- stones. In all of these cases. the gall-bladder bile was invariably inl'ccted with at least one bacterial species which was shown to have active 13- glucuronidase activity. and bile microscopy showed high CaB prccipitation. These results showed significant similarities to thosc observed prel iously with thc brown pigment gallstones. Nanicly. (I) pallstonc nuclear area composition consisting predominantly of CaB and also CaPalm. and (ii) the prcsence in bile of bacterial overgrowth with active fi-glucuronidase and abundant CaB precipitation.

Among thc organisms culturcd from thc gall- bladder biles. thc itiost cominon was E . coli. with the majority of thc othcr bacterial species also being entcric organisms. a result that was consistent with previous work.".'" .'' Bacterial genera observed to have thc active /$glucuronidase capacity were E. (A" K. o.y\focii. B.,li.trCqilis. and c'. per/iriti+wu. similar to the previous lindings by Tabata and Nakayania.

Although the results of this study strongly sug- gest that a group of cholesterol gallstones have a similar nucleating mechanism to the brown pigment gallstones. niarked differences in their overall chemical composition and structure would tend to indicatc otherwise. However. on the basis of the data in the present study. hactcrial overgrowth with active /&glucuronidase. a key factor in the patho- genesis of brown pigment gallstones as postulated by Maki, could aid and accelerate the pathogenic process o f cholesterol gallstones.' That is. i t i s possiblc to suggest that bile. supersaturated with cholcsterol and stored by a gall-bladder that can go on to harbour cholesterol gallstones. could form these gallstones as a direct result of the abundant quantitics of CaR precipitate due to bacterial over- growth specifically with active /%glucuronidasc. This hypothesis was found to correlate with the bile microscopy result5 of 13 patients with multiple cholesterol gallstones. whose corresponding gall- bladder biles were observed to contain abundant aniounts of yellowish-brown CaB precipitatc that wa4 always dispersed diffusely in bilc. cnmeshed

with monohydrate Chc (Fig. 2). I t is almost certain that the CaB precipitate in bile is a most readily available source o f nucleating pigment for the eventual deposition of cholesterol. Having estab- lished this process o f nucleation, cholesterol and brown pigincnt gallstones could then follow diver- gent aetiological paths that correlate with their overall individual morphological and chemical characteristics.

I t should be noted. however, that the frequency of CaB deposits in the centre of the cholesterol gallstones exceeded that o f patients with biles infected with bacterial species having active B- glucuronidase. Moreover. CaB was present in the nuclear areas of cholesterol gallstones, even when the gall-bladder bile was found to be sterile. These observations clearly indicated that CaB formation was not solely dependent on bacterial p- glucuronidasc activity in bile. Indeed. a defect in hepatic bilirubin glucuronidation has been sugges- ted as a cause of increased lcvcls o f insoluble bilirubin in bile and this was postulatcd as an initia- tor of cholesterol gallstonc formation. In Convin- cing evidence. howcvcr. must be obtained from a prospective study. which should include a detailed bacteriological examination of thc gallstones. as well as quantitation of the CaB precipitatc in the cholesterol gallstones and gall-bladder bile. The most recent results o f this study have shown posi- tive bacterial cultures in cholesterol gallstone cen- trcs which wcre comparable with those cultured from the gall-bladder bile (unpubl. data). Thus i t would sceiii that bacteria might be present at the time of cholesterol gallstone nucleation.

The present results. therefore, suggest strongly that bacterial activity contributes to cholesterol gallstone formation. An increased proportion of bilirubin which can precipitate as CaB could be a significant trigger factor for cholesterol gallstone formation at a given saturation o f biliary cholesterol.

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BACTERIA AND GALLSTONE NUCLEATION 577

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