The role of calcium precipitation in the sulfoglycolithocholate-induced cholestasis of the bile fistula hamster
Sulfate glycolithocholic acid (SGLC) has been shown to be highly cholestatic in tlte rat. This study was parformed ia order to gain understanding of the mechanisms of SGLC-indwed chokstasis and ibe aim of the investigation was to explore the hypothesis that SGLC could cawe a precipitation of calcium in bile. We studied the effects of intravenously admitdstnted SGLC on bile flow, biliary lipids secretion and calcium excretion in the female bile fistola hamster. We also performed invitro studies with a CaLI-selective electrode in order to m~asore the calcium binding capacity of SGLC. The results showed that after 1 b of infusion of 8ymoVtW g body weight [“C]SGLC bile ftow dropped to zero. During the infusion period P fine white sludge was visible in the test tube used for bile collection. TLC and HPLC analysis of both tbe superoataut and the precipitate showed that unchanged SGLC wasexcreted into bile. UptoZO% ofbiliary SGLC and more thao 50% ofthe total Ca*+ present in bile was precipitated. The SGLC/C&+ molar ratio in tk precipitate was 1.12 f 0.3 (mean + S.D. of four experiments). Light and electron microscopy of the liver did not show any specific almormalities. The Ca2* binding activity of SGLC in vitro, was highest among the bile acids tested at a concentration of 0.1 mM, when almost 100% of bik (non-micelIar) form. This suggeststhat among the bile acids, SGLC exerts the strongest bittdina activitv an free calciumions. These results confirm the hvwthesis that SGLC mav com:cipitatc with Ca2’ ions itt stoi.
acids are in the mooomeric
or within the caaaliculas.
Sulfate glycolitbocholic acid (SGLC) is one of themetabolites of lithocbolic acid (LCA) present ta normal hu-
understood. It has recently been proposed that the cholestatic action of SGLC is similar to that of another LCA
man bile (1-5). The sulfation of the three hydtoxyl groups
metabolite, LCA-gluwronide (8), and might be related its interaction with calcium either within the liver or in bide canalicutur (9-11). More recently, KuiPws et al. confirmed the cholenatic properties of SGLC end pandcd their observations by demonstrating that the
of LCA has always been proposed as a mechanism of liver detoxification (1-5). SGLC in the monomer form. however, has a poor acqoeoos volubility and a high criti4 micellar temperature, in spite of its low critical micellar coocentration (CMC) (0.5 mM) (6). Yousef et al. (7) demonstrated that SGLC is cholestatic in rat, while the tawine conjugated form. sulfotaurolithocholate @TLC) is not. The mechanism of SGLC-induced cholestasis is poorly
to the (9) exbe-
patotoxiceffectof SGLCis muchmorepronouncedinrats with an exhausted bile acid pool. The possibility tba: SGLC could be cholestatic becauseof its relative water insolubility has also been discussed(10). To date, however,
357 no explanation &IS for the mechanism of SGLC-induced cholestasis. I” this study, we attempt to confirm or excludethe pm. sibility that SGLC-induced cholestasis is caused by precipitation of calciumions. We studied the effect of intravenously administered SGLC o” biliary lipids and calcium excretion in the female hzonster.The hamster was chosen since its bile acids (BA) pool composition and metabolism is similar to that of ma”. We alao comparedthe calciumprecipitation binding capacityof SGLC with other bile acids in vitro, using a Ca*+-selectiveekctmde(12).
Animal
wo’ uperimemtvlproceduret
Female Golden Syria” hamster (W-X&lg body weight, Charles River, CatcaComo. Italy) were used in all axprfments. The fasted animals were anesthetized io the morning with an intraperitoneal injectioe of 0.1 mUlO g b. wt. of 2.5 rag/ml of xflaaine, and MImglml of ketamine hydroehlorfdc. The femoral vein was then isolated and ca”““lated with a PE 10 catlreter (clay Adams, NI. U.S.A.), and a solution of 0.9 B/la, “II NaCl was infused a, a Uov, rate of 1 mb’h,using a 8. Braun (Mclsungen, F&G.) infvsfo” pump. Dmiog the e”1i.e duration of the oxperimeot, body temperature war monitored and maintained at 37 ‘C with a beating lamp. After chokcfstectomy. the bile duct was cannolated with a PE 10 catheter, and bile was collected at 10 min intervalp for 30 min. After tbc bile flow became regular, an intiion of 8 ~mol/l@l g body u?.m of SGLC (Calbi &em, La Jolla, CA, U.S.A.), wntaining 50 “Ci of [“qSGLC (a kind gift of Dr. F. Kuipers), dissolved in oomtal saline containing 7.5% of albumin, was started. Thereaher, bii ozdketionwas prolonged for 1 h. Bile oolkctfon was not extended beyond 100 min becaw we wa”ted to keep the bile acidpoolfntact, asit haspreviaw ly been demonstrated (13,14) that at least 4 h are neeeswy to reach 50% depletfon of BA pool (14) using the same bile Mtula hamster model. After can”“lation of the main bile duct, bile flow was measured gravimenically by colkctfng bile at 10 min inter&s for 30 mh~in preweighed test tubes before SGLC infusion. During SGLC infusion, a fine white precipitate was alwaysvisible withii the bidein the tubes. Aftn the fast four pilot expcrimcnts. in order to obtain all the bile secreted and the precipitate formed during SGLC infosioo one tobe per hamster was used for bile mllectfo” instead of changf”gsix different tubes every 10mie. After I h, the tube was weighed and the bile was centrifoged at
3ooOX g for 10 min. The transparent. fresh white bile in the supematanr w= transferred to another tube, and the white precipitate was washed hvice with iodized water, centrifugedand finally resuspendedin 1 ml of methanol.
Aliquots of 10~1of bile and lOOpIof the metbanol-dissolved precipitate were dried out, extracted in methanol and Counted in a Becltman LS 1701 beta-scintillation coanter quench corrected by the channel ratio method using a” external standard after adding 10 ml of lostage (United PackardTechhn.,Dower Growers. IL, U.S.A.). All the semplesleft were maintained at -80 T until use. The wnceetratioo of bile acids, cholesterol snd phospholipids in the bile samples was measured using pm v&sly describedenzymaticmethods (15-17). [“CISGLC purity waschecked by both HPLCand TLC (18-19) and was alwaysmore than 98%. SGLC and other individual conjugated bile aciL ia the bik were analyzed by HPLC on an LC SW0Varia” instmment (Varfan,Palo Alto. CA. U.S.A.), a Vtia” column heater and a Vwian CDS 1llL area integrator, using a” Rp-18, Spheri-5 cnlurn”. 4.6 mm x 250 mm (Bmwnce Labs Inc., Santa Clara, CA, U.S.A.), according to the methad of Nakayama and Nakagaki (18). Bile sampler were extracted with 10 volr. of methanol: the sample was dried, resuspended in the solvent system (acetonirrile/methanollpotassium phosphate buffer. 3U mM (pH 3.40) 10:60:30 (v/v) and filtered tbrougb a disposable Millipore HV filter, pore size
0.45 pm. Testortetoae acetate was used as internal standard. The tlow rate was1.0 m&n with2000 p.s.i. isobaric flow. Detection was made at 205 “m and the tempera. tore was kept at 40 ‘c. Uoder these conditions, SGLC recoveps WK more than 98% and all the conjugated bile acids wereeluted within 40-45 min. The presence of SGLC in the precipitate and in the s”pematant was checked both by HPLC, using the same method describedalwe, and TLC accordingto the method described by Cass et al. (19). The solvent system uSed for TLC was a solution of chloroformlmethanoUacetic acidiwater 65:24:15:9 (V/V).Twenty pl sa”lpks were plated on precoated silk acid ewJ calcium sulfate (10%) plates underco”ditiunsofchamber~Nradon. Total calcium in bik collected before SGLC infusion in the sopmtataot and in the precipitate was measured by pblamephotometry.
Solutions were all prepared in glass tubes and analyzed in duplicate o” the Danteday, as previously desmiid (201. Four CaCl2 standard solutions (0.01, 1.0 and 10 mM) in 0.1 M Na-barbital boffer (pH 8.8) were prepared.
S. BELLENTANI c, al.
353
NsCl was added to each solution in order to stabilize the ionic strength to I = 0.16; an appropriate amount of so-
s1atisticl?/amtlysis The results are expressed
crose was used to give a standard osmolsrity of 300 mOsmlkg. §tockCaCi~solutions were kept under paraffin oil after bubbling wifh N,. Four different solutions of various bile acids were prepared; commercial bile acids were dialyzed in the Presence of EDTA for 24 h before starting the experiments. A final volume of 30 ml was reached by addition of double ionized water (resistivity = 18
significance was evaluated
Rdt5
MOhm/cm-‘1. For SGLC, only two concentrations were obtained (0.1 and 1.0 mM), because at concentrations higher than 1.0 mM solubility was poor and solutions had a milky appearante Ionized calcium was determined using the Caa+-selective electrode in duplicate at 25 *C and at 37 “c (when
tervals. Before SGLC infusion, bile flow was constant and reached values similar to those previously found in the female hamster (13). A few minutes after the begianing infusion, the bile flow started to drop and afine white sludge was visible in the PElO catheter and in the colIection tubes. Bile flow dropped to zero within 60 min after the beginning of the SGLC infusion.
SGLC was used) with an EA 940 Expandable Ionanalyzer obtained from Orion Research Inc. (Boston, MA. U.S.A.). All scJutians were warmed to avoid drifting due 10 continuous stirring. Usually, the electrode potential is a function of Ca*+ activity, thus the potential mt-asured (E) reflects the number of Ck2+ ionr present in solution. The potential (E) can be obtained from the Nemst equation:
Measurement of bile acids (BA), phospholipids and cholesterol found in the pool of bii collected duriog the 30 mln before and the M min after the beginning of SGLC infusion is reported in Tabte 1. In the bile collected after SGLC injection, the total BA and chotesterol contmt was sigoificantly lower than that found before SGLC infusioo. Instead, phospholipid Identical to hasal values, indicating a greater secretion of phospbollpId than of BA and cholesterol during SGLC infusion. Both light and electron miaoscopy of the liver tiasoe fromSG~-injectedhamstcnshowcdai~epat~struv ture within normal limits. AU cytoplannic orgaoelles ap
E = E + 2.3 RThFlog(A) where: E is the standard potential of the system; R is the universal constant of gas; T is the absolute temperature; R is the number of the electrons; F is the charge associated with 1 mol of electrons (Faraday); and A is the activity coefficient of the anion or cation that moat be measured. Since the aim of this work was the evaluation
of free
Ca*’ available for binding, the ionic strength was kept constant by adding an adequate amount of NaCl to each
as mean
+ S.D. Statistical
by Student’s unpaired t-test.
Fig. 1 shows the effect of SGLC infusion on bile flow io the first four animals, whenbile
was collected at 10 min in-
content was alnmt
peared normal, with the exception of a swelling of the mltochondria (Fig. 1). Bile canaliculi were normal and Iined by regular microvilll. The only peculiar change in the liver of SGLC-infused
animals was the presence
of small, uo-
identified dense bodies, simitar to those found by Miyai et al. (Zl), in the pericaordicular region, mainly inside the
SOlUtiOll. In this study, standard calibration curves showed that the electrode response was linear over a 10m5to lo-’ M
concentration
range.
At the end of the experiment, the liver was removed from three animals for examination by light and electron microscopy (EM). Smalls&ions (l-mm cubes) from each of the lobes of the liver were cut and fixed either in formaldehyde, for light microcopy, or in Millonig’a osmium fixative for I h at 4 ‘C, for EM. Specimens for EM were dehydrated in graded ethanol and embedded in Epon. These seclions were cut and stained with uranyl acetate and lead citrate. Morphologic evaluation was made by a separate investigator who bad no knowledge of the experimental protocol.
tlmelmhl b. I. Bile flow in bile firrelahamstert(ewe f S.D. of feure*pcrfmerits) before md after infusion of B~melfloOg per h of SGLC. The basal bile collection lasted 30 min. The arrow indicates lb2 be. ginning nf the inhim (32ndmin).
Ce
TABLE
1
granuhxitic or the Kuppfer cells around the sinusoidal region (see Figs. 2 and 3, precipitates? degradation material?). The total amount of calcium found in gallbladder (0.147 i 0.015 mg/ml, mean + SD.) and hepatie (0.083 f 0.003) bii before the injection of SGLC was sintihu to that found in control animals (data not shown) and within the same range reported by other autltors for the rat (22.23). When we analyzed the total calcium and [l’c]SGLC content in both the supcmatant and the precipitate of bile co&ted after infusioa, we found that the
CaZ’ content in she two fractions was very similar (Table
2). On the contrar)r, the concentration of SGLC present in the bile supematant was almost IO-times greater tbao that found in the precipitate. Furtbet’more, the SGLUc&ium molar ratio in the precipitale was &se to 1 (see Table 2). HPLC analysis of the Me colkcted before SGLC infusian revealed a compaition of bile asids similar to that in c.mtrol animals (24) (data not shown). The supematant of the bik after SGLC infusion, &a siwwed a bite acid corn@tion similar to control animals, except that another
peak, corresponding
to SGLC, was present
in the curve.
On the other hand, as shown in Fig. 4, HPLC analysis of bile add content in the precipitate revealed only one peak, corresponding
to the elution time of SGLC. All the
radirxctivity recovered in the precipitate cotresponded to the peak. Finally, TLC analysis ofthe precipitate showed one spot corresponding to SGLC. The in vitro studies with the calcium electrode showed that the calcium binding activity of SGLC was the highest among all the physiological bile acids tested (twoand glycaconjugated CA, CDCA, LCA and UDCA), at the same concentration of 0.1 mM when almost 100% of bile species are in the monomeric (nonmicellar) form. In Fig. 5 the SGLC calcium activity is compared with three of the bile acids tested (glycochenodeoxycholate, glycodeoxy cholate and taurocholate). The SGLC calcium biding actinty is significantly higher than that of other bile acids, supgesting that SGLCis the bile acid which binds bee calcium ions with fhe mm effciency. Furthermore, within the range of SGLC concentrations explored (NJ-* to IO-’
M), we were not able to find a caIcium concentration that saturates binding before precipitation occurs, thus sug. gestiveof a 1:l stoicbiometry.
361
:o those found by Miyai et al. (21). which we found inside he Koppfer cells around the sinusoidal region. The rerults obtained from the analysis of the precipitates found within the bile during SGLC infusion showed that uomodified SGLC was secx;eZ i; bile all the, up 20% o: i. precipitates together with 4550% of the blliary C&’ ions. The calcium-selective electrode rxperiments showed that SGLC was the B.4 with she ixgbest Ca*+ binding activity among the physiological bile acids I 0.1 mM concentration when the molecules are present in a nonmicellar, monomeric form. Furthermore, within the range of SGLC concentrations explored (lo-’ to IO-* M), we were not able to find a calcium concentration that saw rates the binding (es happens for othex BA) before predp i-ation occurred. The curve (see Fig. 5) suggests that SSLC binds calcium ions in a 1:l stoichiomeky. Gne par-
ia
sible explanation d ali these phenomena is that when a critical SGLC volubility concentration is reached in bile (around 10 mM), one molecule of SGLC, by chelating one molecule of C&, turns its physical state boom a liquid-soluble into a crystal-insoluble form. The Sigh Calcium binding properties end the linearity of the binding in the in vitro experiments suggest that both the carboxy and the sulfate gxooups of SGLC are involved in the binding of calcium. Other authors (31,32) have previously reported that some cholestatic bile acids, such as LCA and especially the sulfate or +eronide derivatives, sulpbolitbocbolate (LCS) and lithocholic acid glumronide (LCG), being pea mklle formas (31). bind calcium I& Wtimes more effectively than taupciiolate (TC) or gly. oxholate (GC). The data reported in this paper confirm that previous findiig obtained in the rat (7-10) also pertain to the female hamster
SGLC is highly cholestatic
duced cl&stasis
and SGLC-in-
may be due to precipitation
of an insol-
uble form of SGLC and W+ ion aggregate:. After 1 h of infusion of SGLC, bile flow dropped to zero and a fine white sludge precipitate was visible in the collected bile. The critical cholestatic dose (B/rmoUl@l g body wt. per h) is lower than that found by You& et al. in the rat (24 rmoI/lODgbodyw.)
(7). Tbisisperhaps
due to thediffer-
ent composition of BA in the two species (l&24,25), thus leading to a different cakium binding capacity of the bile. The conditions found in tbe hamster were felr to be ctoser to those in humans, due to similarities of biliary BA pool composition and of cholesterol and BA synthesis in man andthe hamster (13.24). Light and electron microscopy examination of liver tissue taken from SGLC-infused hamsters did not show any specific alterations. The only linding which could resemble SGLC precipitates are the small dense bodies, similar
Kuipers et al. recently demonstrated that the intestinal absorption of SGLC is greatly effected by the presence of an excess of calcium ions in the intestinal lumen (IO) and they speculated that the reduced absorption may be the result of precipitation of SGLC as calcium salts (10). Another interesting point of OUTresults is that during SGLC intikn. in mite of a reduction of BA and cholesterol secretion, phospholipid seaetion remained unchanged in respect to basal values. Tbis particular effect of SGLC on biliary lipid secretion has, to our knowledge, never been described before. It is well known that the biliery secretion of most BA induces the secretion of pbospholipids and cholesterol into bile (26-28) and that the lower the CMC afabile acid, the grrarer isitsinducingeffeet on lipid secretion (6). SGLC, as mentioned above, has paor solubilii and a low CMC, easily inducing biliary lipid secretion. The of lipid secretion from BA and cholesterol secretion by SGLC is more difficult to explain. If rbis is confirmed by other studies, however, it could have serious impliitione for the cwreet theory that bile salts and phospbolipidkholesterol vesicles are se-
uncoupling
s. BELLENTANI
362
creted into the bile independently
aggregating
thereafter
to form micelles. Kuipen et al. found that SGLC inhibits biliarv ohosoholioids and cholesterol secretion in the rat. but only when administered in relatiwly low, nan.choles
,.
.
tatic doses (29,30). Another explanation for this result might be that SGLC awegates with bile acid/phosphatidylcholine mixed micelles which reduces the cholesterol solubilizing capacity. This has been previously demonstrated in vitro by Carey et al. (6). So far, this interesting and unexpected uncoupling phenomenon remains an isolated obswvaticz and whether it OCCURat the canalicular level or inside the hepatocyte, is not possible to determine from our experiments. We conclude that our data together with other observations (10-12) suggest that the pathogenesis of cholestasis induced by SGLC or LCG, may be related to their greater affinity for calcium ions and the formation of insoluble calcium salts. Presently, data reported can not distinguish the level at which the complex SGLC-calcium aggregates inside the hepatocyte (for example inside the micmtu-
cd&m. Gastroentcmlogy1975. 6 CarcgMC, Small DM, Bliss CM. Lipid digestionand admptio”. Ann Rev Physiol,983;45: 651-677. 7 Youret IM, Tucbwekr B, “ank Ft.!, Masse D. Audet M, Roy CC. Lithochoiute cbolestash-sulfated&co,idmho,ate induced intrabepadc chalestasis in rats. Gartmpnterology 1981; 80: 233-241. 8 Gelberg DG. Chad MV. Little JM, Adcock EW and Lener R. Lirbaeholaw glucuranide is a cholestadc agent. J Cl,” Invest 19m73: mlFlS,4. 9 Ruipers F. Havinga R, Vonk Iw. Cbolestasisinduced by sulfate giycdilbocholic atid in Aberal: protection by endagenousbib acid.. Cli” Sci Land 1985.68: 127.134. 10 K”ipers F, Halinga H. Havinga R, “0nk RI. t”tesd”al absarp donollilbocholic acidsulfatssi” the rat: inbibitoryelfectat c&i. urn. Am J Physic, 1986; 251: (Gartroinrcst Liver Pbysiol 14):
et a,.
of C$+ is high) or within the catnliculus. In turn, the SGLC-induced cholestasis could be the result of a reduction cd biliarv free calcium concentration, thus of a disturbance of thk intracellular bulcs. where the conwmtration
transport system and/or of the function of the tight junctions (11,33,34), or only a mechanical obstrwticm due to a time and rate-dependent accumulation of SGLC-calcium crystals within the bile ducts. The unspecificity of the mmphological alterations found at EM and the integrity of the livercell support the latter hypothesis.
.4cknowledgEnlMt We thank our friend Dr. Folkert Kuipers for supplying us with the [‘4C]sulfoglyeotithoholate. This work was funded by a CNR Grant No. g7.01543.C4 and by private Grants of the Fonda per lo Studio deUe M&tie to.
de1 Fega-
18 NsksysmaF. Nakagaki M. Quantitative determination of bits acUs in bile with reversedphw high pcrfammce liquid cbrc. mat,grspby. J chramatogJ ,980: 18: 267-293. 19 cauow, Cmve” AE. “obna”” AF,Cc& SB. Tld”.,ayeycrcbro. “mtwwhk warado” ot rulfaed and m”s,dfa,cd ,i,hasho,ic
Sulfate glycolithocholic acid (SGLC) has been shown to be highly cholestatic in tlte rat. This study was parformed ia order to gain understanding of the mechanisms of SGLC-indwed chokstasis and ibe aim of the investigation was to explore the hypothesis that SGLC could cawe a precipitation of calcium in bile. We studied the effects of intravenously admitdstnted SGLC on bile flow, biliary lipids secretion and calcium excretion in the female bile fistola hamster. We also performed invitro studies with a CaLI-selective electrode in order to m~asore the calcium binding capacity of SGLC. The results showed that after 1 b of infusion of 8ymoVtW g body weight [“C]SGLC bile ftow dropped to zero. During the infusion period P fine white sludge was visible in the test tube used for bile collection. TLC and HPLC analysis of both tbe superoataut and the precipitate showed that unchanged SGLC wasexcreted into bile. UptoZO% ofbiliary SGLC and more thao 50% ofthe total Ca*+ present in bile was precipitated. The SGLC/C&+ molar ratio in tk precipitate was 1.12 f 0.3 (mean + S.D. of four experiments). Light and electron microscopy of the liver did not show any specific almormalities. The Ca2* binding activity of SGLC in vitro, was highest among the bile acids tested at a concentration of 0.1 mM, when almost 100% of bik (non-micelIar) form. This suggeststhat among the bile acids, SGLC exerts the strongest bittdina activitv an free calciumions. These results confirm the hvwthesis that SGLC mav com:cipitatc with Ca2’ ions itt stoi.
acids are in the mooomeric
or within the caaaliculas.
Sulfate glycolitbocholic acid (SGLC) is one of themetabolites of lithocbolic acid (LCA) present ta normal hu-
understood. It has recently been proposed that the cholestatic action of SGLC is similar to that of another LCA
man bile (1-5). The sulfation of the three hydtoxyl groups
metabolite, LCA-gluwronide (8), and might be related its interaction with calcium either within the liver or in bide canalicutur (9-11). More recently, KuiPws et al. confirmed the cholenatic properties of SGLC end pandcd their observations by demonstrating that the
of LCA has always been proposed as a mechanism of liver detoxification (1-5). SGLC in the monomer form. however, has a poor acqoeoos volubility and a high criti4 micellar temperature, in spite of its low critical micellar coocentration (CMC) (0.5 mM) (6). Yousef et al. (7) demonstrated that SGLC is cholestatic in rat, while the tawine conjugated form. sulfotaurolithocholate @TLC) is not. The mechanism of SGLC-induced cholestasis is poorly
to the (9) exbe-
patotoxiceffectof SGLCis muchmorepronouncedinrats with an exhausted bile acid pool. The possibility tba: SGLC could be cholestatic becauseof its relative water insolubility has also been discussed(10). To date, however,
357 no explanation &IS for the mechanism of SGLC-induced cholestasis. I” this study, we attempt to confirm or excludethe pm. sibility that SGLC-induced cholestasis is caused by precipitation of calciumions. We studied the effect of intravenously administered SGLC o” biliary lipids and calcium excretion in the female hzonster.The hamster was chosen since its bile acids (BA) pool composition and metabolism is similar to that of ma”. We alao comparedthe calciumprecipitation binding capacityof SGLC with other bile acids in vitro, using a Ca*+-selectiveekctmde(12).
Animal
wo’ uperimemtvlproceduret
Female Golden Syria” hamster (W-X&lg body weight, Charles River, CatcaComo. Italy) were used in all axprfments. The fasted animals were anesthetized io the morning with an intraperitoneal injectioe of 0.1 mUlO g b. wt. of 2.5 rag/ml of xflaaine, and MImglml of ketamine hydroehlorfdc. The femoral vein was then isolated and ca”““lated with a PE 10 catlreter (clay Adams, NI. U.S.A.), and a solution of 0.9 B/la, “II NaCl was infused a, a Uov, rate of 1 mb’h,using a 8. Braun (Mclsungen, F&G.) infvsfo” pump. Dmiog the e”1i.e duration of the oxperimeot, body temperature war monitored and maintained at 37 ‘C with a beating lamp. After chokcfstectomy. the bile duct was cannolated with a PE 10 catheter, and bile was collected at 10 min intervalp for 30 min. After tbc bile flow became regular, an intiion of 8 ~mol/l@l g body u?.m of SGLC (Calbi &em, La Jolla, CA, U.S.A.), wntaining 50 “Ci of [“qSGLC (a kind gift of Dr. F. Kuipers), dissolved in oomtal saline containing 7.5% of albumin, was started. Thereaher, bii ozdketionwas prolonged for 1 h. Bile oolkctfon was not extended beyond 100 min becaw we wa”ted to keep the bile acidpoolfntact, asit haspreviaw ly been demonstrated (13,14) that at least 4 h are neeeswy to reach 50% depletfon of BA pool (14) using the same bile Mtula hamster model. After can”“lation of the main bile duct, bile flow was measured gravimenically by colkctfng bile at 10 min inter&s for 30 mh~in preweighed test tubes before SGLC infusion. During SGLC infusion, a fine white precipitate was alwaysvisible withii the bidein the tubes. Aftn the fast four pilot expcrimcnts. in order to obtain all the bile secreted and the precipitate formed during SGLC infosioo one tobe per hamster was used for bile mllectfo” instead of changf”gsix different tubes every 10mie. After I h, the tube was weighed and the bile was centrifoged at
3ooOX g for 10 min. The transparent. fresh white bile in the supematanr w= transferred to another tube, and the white precipitate was washed hvice with iodized water, centrifugedand finally resuspendedin 1 ml of methanol.
Aliquots of 10~1of bile and lOOpIof the metbanol-dissolved precipitate were dried out, extracted in methanol and Counted in a Becltman LS 1701 beta-scintillation coanter quench corrected by the channel ratio method using a” external standard after adding 10 ml of lostage (United PackardTechhn.,Dower Growers. IL, U.S.A.). All the semplesleft were maintained at -80 T until use. The wnceetratioo of bile acids, cholesterol snd phospholipids in the bile samples was measured using pm v&sly describedenzymaticmethods (15-17). [“CISGLC purity waschecked by both HPLCand TLC (18-19) and was alwaysmore than 98%. SGLC and other individual conjugated bile aciL ia the bik were analyzed by HPLC on an LC SW0Varia” instmment (Varfan,Palo Alto. CA. U.S.A.), a Vtia” column heater and a Vwian CDS 1llL area integrator, using a” Rp-18, Spheri-5 cnlurn”. 4.6 mm x 250 mm (Bmwnce Labs Inc., Santa Clara, CA, U.S.A.), according to the methad of Nakayama and Nakagaki (18). Bile sampler were extracted with 10 volr. of methanol: the sample was dried, resuspended in the solvent system (acetonirrile/methanollpotassium phosphate buffer. 3U mM (pH 3.40) 10:60:30 (v/v) and filtered tbrougb a disposable Millipore HV filter, pore size
0.45 pm. Testortetoae acetate was used as internal standard. The tlow rate was1.0 m&n with2000 p.s.i. isobaric flow. Detection was made at 205 “m and the tempera. tore was kept at 40 ‘c. Uoder these conditions, SGLC recoveps WK more than 98% and all the conjugated bile acids wereeluted within 40-45 min. The presence of SGLC in the precipitate and in the s”pematant was checked both by HPLC, using the same method describedalwe, and TLC accordingto the method described by Cass et al. (19). The solvent system uSed for TLC was a solution of chloroformlmethanoUacetic acidiwater 65:24:15:9 (V/V).Twenty pl sa”lpks were plated on precoated silk acid ewJ calcium sulfate (10%) plates underco”ditiunsofchamber~Nradon. Total calcium in bik collected before SGLC infusion in the sopmtataot and in the precipitate was measured by pblamephotometry.
Solutions were all prepared in glass tubes and analyzed in duplicate o” the Danteday, as previously desmiid (201. Four CaCl2 standard solutions (0.01, 1.0 and 10 mM) in 0.1 M Na-barbital boffer (pH 8.8) were prepared.
S. BELLENTANI c, al.
353
NsCl was added to each solution in order to stabilize the ionic strength to I = 0.16; an appropriate amount of so-
s1atisticl?/amtlysis The results are expressed
crose was used to give a standard osmolsrity of 300 mOsmlkg. §tockCaCi~solutions were kept under paraffin oil after bubbling wifh N,. Four different solutions of various bile acids were prepared; commercial bile acids were dialyzed in the Presence of EDTA for 24 h before starting the experiments. A final volume of 30 ml was reached by addition of double ionized water (resistivity = 18
significance was evaluated
Rdt5
MOhm/cm-‘1. For SGLC, only two concentrations were obtained (0.1 and 1.0 mM), because at concentrations higher than 1.0 mM solubility was poor and solutions had a milky appearante Ionized calcium was determined using the Caa+-selective electrode in duplicate at 25 *C and at 37 “c (when
tervals. Before SGLC infusion, bile flow was constant and reached values similar to those previously found in the female hamster (13). A few minutes after the begianing infusion, the bile flow started to drop and afine white sludge was visible in the PElO catheter and in the colIection tubes. Bile flow dropped to zero within 60 min after the beginning of the SGLC infusion.
SGLC was used) with an EA 940 Expandable Ionanalyzer obtained from Orion Research Inc. (Boston, MA. U.S.A.). All scJutians were warmed to avoid drifting due 10 continuous stirring. Usually, the electrode potential is a function of Ca*+ activity, thus the potential mt-asured (E) reflects the number of Ck2+ ionr present in solution. The potential (E) can be obtained from the Nemst equation:
Measurement of bile acids (BA), phospholipids and cholesterol found in the pool of bii collected duriog the 30 mln before and the M min after the beginning of SGLC infusion is reported in Tabte 1. In the bile collected after SGLC injection, the total BA and chotesterol contmt was sigoificantly lower than that found before SGLC infusioo. Instead, phospholipid Identical to hasal values, indicating a greater secretion of phospbollpId than of BA and cholesterol during SGLC infusion. Both light and electron miaoscopy of the liver tiasoe fromSG~-injectedhamstcnshowcdai~epat~struv ture within normal limits. AU cytoplannic orgaoelles ap
E = E + 2.3 RThFlog(A) where: E is the standard potential of the system; R is the universal constant of gas; T is the absolute temperature; R is the number of the electrons; F is the charge associated with 1 mol of electrons (Faraday); and A is the activity coefficient of the anion or cation that moat be measured. Since the aim of this work was the evaluation
of free
Ca*’ available for binding, the ionic strength was kept constant by adding an adequate amount of NaCl to each
as mean
+ S.D. Statistical
by Student’s unpaired t-test.
Fig. 1 shows the effect of SGLC infusion on bile flow io the first four animals, whenbile
was collected at 10 min in-
content was alnmt
peared normal, with the exception of a swelling of the mltochondria (Fig. 1). Bile canaliculi were normal and Iined by regular microvilll. The only peculiar change in the liver of SGLC-infused
animals was the presence
of small, uo-
identified dense bodies, simitar to those found by Miyai et al. (Zl), in the pericaordicular region, mainly inside the
SOlUtiOll. In this study, standard calibration curves showed that the electrode response was linear over a 10m5to lo-’ M
concentration
range.
At the end of the experiment, the liver was removed from three animals for examination by light and electron microscopy (EM). Smalls&ions (l-mm cubes) from each of the lobes of the liver were cut and fixed either in formaldehyde, for light microcopy, or in Millonig’a osmium fixative for I h at 4 ‘C, for EM. Specimens for EM were dehydrated in graded ethanol and embedded in Epon. These seclions were cut and stained with uranyl acetate and lead citrate. Morphologic evaluation was made by a separate investigator who bad no knowledge of the experimental protocol.
tlmelmhl b. I. Bile flow in bile firrelahamstert(ewe f S.D. of feure*pcrfmerits) before md after infusion of B~melfloOg per h of SGLC. The basal bile collection lasted 30 min. The arrow indicates lb2 be. ginning nf the inhim (32ndmin).
Ce
TABLE
1
granuhxitic or the Kuppfer cells around the sinusoidal region (see Figs. 2 and 3, precipitates? degradation material?). The total amount of calcium found in gallbladder (0.147 i 0.015 mg/ml, mean + SD.) and hepatie (0.083 f 0.003) bii before the injection of SGLC was sintihu to that found in control animals (data not shown) and within the same range reported by other autltors for the rat (22.23). When we analyzed the total calcium and [l’c]SGLC content in both the supcmatant and the precipitate of bile co&ted after infusioa, we found that the
CaZ’ content in she two fractions was very similar (Table
2). On the contrar)r, the concentration of SGLC present in the bile supematant was almost IO-times greater tbao that found in the precipitate. Furtbet’more, the SGLUc&ium molar ratio in the precipitale was &se to 1 (see Table 2). HPLC analysis of the Me colkcted before SGLC infusian revealed a compaition of bile asids similar to that in c.mtrol animals (24) (data not shown). The supematant of the bik after SGLC infusion, &a siwwed a bite acid corn@tion similar to control animals, except that another
peak, corresponding
to SGLC, was present
in the curve.
On the other hand, as shown in Fig. 4, HPLC analysis of bile add content in the precipitate revealed only one peak, corresponding
to the elution time of SGLC. All the
radirxctivity recovered in the precipitate cotresponded to the peak. Finally, TLC analysis ofthe precipitate showed one spot corresponding to SGLC. The in vitro studies with the calcium electrode showed that the calcium binding activity of SGLC was the highest among all the physiological bile acids tested (twoand glycaconjugated CA, CDCA, LCA and UDCA), at the same concentration of 0.1 mM when almost 100% of bile species are in the monomeric (nonmicellar) form. In Fig. 5 the SGLC calcium activity is compared with three of the bile acids tested (glycochenodeoxycholate, glycodeoxy cholate and taurocholate). The SGLC calcium biding actinty is significantly higher than that of other bile acids, supgesting that SGLCis the bile acid which binds bee calcium ions with fhe mm effciency. Furthermore, within the range of SGLC concentrations explored (NJ-* to IO-’
M), we were not able to find a caIcium concentration that saturates binding before precipitation occurs, thus sug. gestiveof a 1:l stoicbiometry.
361
:o those found by Miyai et al. (21). which we found inside he Koppfer cells around the sinusoidal region. The rerults obtained from the analysis of the precipitates found within the bile during SGLC infusion showed that uomodified SGLC was secx;eZ i; bile all the, up 20% o: i. precipitates together with 4550% of the blliary C&’ ions. The calcium-selective electrode rxperiments showed that SGLC was the B.4 with she ixgbest Ca*+ binding activity among the physiological bile acids I 0.1 mM concentration when the molecules are present in a nonmicellar, monomeric form. Furthermore, within the range of SGLC concentrations explored (lo-’ to IO-* M), we were not able to find a calcium concentration that saw rates the binding (es happens for othex BA) before predp i-ation occurred. The curve (see Fig. 5) suggests that SSLC binds calcium ions in a 1:l stoichiomeky. Gne par-
ia
sible explanation d ali these phenomena is that when a critical SGLC volubility concentration is reached in bile (around 10 mM), one molecule of SGLC, by chelating one molecule of C&, turns its physical state boom a liquid-soluble into a crystal-insoluble form. The Sigh Calcium binding properties end the linearity of the binding in the in vitro experiments suggest that both the carboxy and the sulfate gxooups of SGLC are involved in the binding of calcium. Other authors (31,32) have previously reported that some cholestatic bile acids, such as LCA and especially the sulfate or +eronide derivatives, sulpbolitbocbolate (LCS) and lithocholic acid glumronide (LCG), being pea mklle formas (31). bind calcium I& Wtimes more effectively than taupciiolate (TC) or gly. oxholate (GC). The data reported in this paper confirm that previous findiig obtained in the rat (7-10) also pertain to the female hamster
SGLC is highly cholestatic
duced cl&stasis
and SGLC-in-
may be due to precipitation
of an insol-
uble form of SGLC and W+ ion aggregate:. After 1 h of infusion of SGLC, bile flow dropped to zero and a fine white sludge precipitate was visible in the collected bile. The critical cholestatic dose (B/rmoUl@l g body wt. per h) is lower than that found by You& et al. in the rat (24 rmoI/lODgbodyw.)
(7). Tbisisperhaps
due to thediffer-
ent composition of BA in the two species (l&24,25), thus leading to a different cakium binding capacity of the bile. The conditions found in tbe hamster were felr to be ctoser to those in humans, due to similarities of biliary BA pool composition and of cholesterol and BA synthesis in man andthe hamster (13.24). Light and electron microscopy examination of liver tissue taken from SGLC-infused hamsters did not show any specific alterations. The only linding which could resemble SGLC precipitates are the small dense bodies, similar
Kuipers et al. recently demonstrated that the intestinal absorption of SGLC is greatly effected by the presence of an excess of calcium ions in the intestinal lumen (IO) and they speculated that the reduced absorption may be the result of precipitation of SGLC as calcium salts (10). Another interesting point of OUTresults is that during SGLC intikn. in mite of a reduction of BA and cholesterol secretion, phospholipid seaetion remained unchanged in respect to basal values. Tbis particular effect of SGLC on biliary lipid secretion has, to our knowledge, never been described before. It is well known that the biliery secretion of most BA induces the secretion of pbospholipids and cholesterol into bile (26-28) and that the lower the CMC afabile acid, the grrarer isitsinducingeffeet on lipid secretion (6). SGLC, as mentioned above, has paor solubilii and a low CMC, easily inducing biliary lipid secretion. The of lipid secretion from BA and cholesterol secretion by SGLC is more difficult to explain. If rbis is confirmed by other studies, however, it could have serious impliitione for the cwreet theory that bile salts and phospbolipidkholesterol vesicles are se-
uncoupling
s. BELLENTANI
362
creted into the bile independently
aggregating
thereafter
to form micelles. Kuipen et al. found that SGLC inhibits biliarv ohosoholioids and cholesterol secretion in the rat. but only when administered in relatiwly low, nan.choles
,.
.
tatic doses (29,30). Another explanation for this result might be that SGLC awegates with bile acid/phosphatidylcholine mixed micelles which reduces the cholesterol solubilizing capacity. This has been previously demonstrated in vitro by Carey et al. (6). So far, this interesting and unexpected uncoupling phenomenon remains an isolated obswvaticz and whether it OCCURat the canalicular level or inside the hepatocyte, is not possible to determine from our experiments. We conclude that our data together with other observations (10-12) suggest that the pathogenesis of cholestasis induced by SGLC or LCG, may be related to their greater affinity for calcium ions and the formation of insoluble calcium salts. Presently, data reported can not distinguish the level at which the complex SGLC-calcium aggregates inside the hepatocyte (for example inside the micmtu-
cd&m. Gastroentcmlogy1975. 6 CarcgMC, Small DM, Bliss CM. Lipid digestionand admptio”. Ann Rev Physiol,983;45: 651-677. 7 Youret IM, Tucbwekr B, “ank Ft.!, Masse D. Audet M, Roy CC. Lithochoiute cbolestash-sulfated&co,idmho,ate induced intrabepadc chalestasis in rats. Gartmpnterology 1981; 80: 233-241. 8 Gelberg DG. Chad MV. Little JM, Adcock EW and Lener R. Lirbaeholaw glucuranide is a cholestadc agent. J Cl,” Invest 19m73: mlFlS,4. 9 Ruipers F. Havinga R, Vonk Iw. Cbolestasisinduced by sulfate giycdilbocholic atid in Aberal: protection by endagenousbib acid.. Cli” Sci Land 1985.68: 127.134. 10 K”ipers F, Halinga H. Havinga R, “0nk RI. t”tesd”al absarp donollilbocholic acidsulfatssi” the rat: inbibitoryelfectat c&i. urn. Am J Physic, 1986; 251: (Gartroinrcst Liver Pbysiol 14):
et a,.
of C$+ is high) or within the catnliculus. In turn, the SGLC-induced cholestasis could be the result of a reduction cd biliarv free calcium concentration, thus of a disturbance of thk intracellular bulcs. where the conwmtration
transport system and/or of the function of the tight junctions (11,33,34), or only a mechanical obstrwticm due to a time and rate-dependent accumulation of SGLC-calcium crystals within the bile ducts. The unspecificity of the mmphological alterations found at EM and the integrity of the livercell support the latter hypothesis.
.4cknowledgEnlMt We thank our friend Dr. Folkert Kuipers for supplying us with the [‘4C]sulfoglyeotithoholate. This work was funded by a CNR Grant No. g7.01543.C4 and by private Grants of the Fonda per lo Studio deUe M&tie to.
de1 Fega-
18 NsksysmaF. Nakagaki M. Quantitative determination of bits acUs in bile with reversedphw high pcrfammce liquid cbrc. mat,grspby. J chramatogJ ,980: 18: 267-293. 19 cauow, Cmve” AE. “obna”” AF,Cc& SB. Tld”.,ayeycrcbro. “mtwwhk warado” ot rulfaed and m”s,dfa,cd ,i,hasho,ic
