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Tauroursodeoxycholate prevents taurolithocholate-induced cholestasis and toxicity in rat liver
Tauroursodeoxycholate
prevents taurolithocholate-induced and toxicity in rat liver*
cholestasis
Ursodeoxycholate has been advocated for the treatment of cholestatic liver diseases. The coinfusion of taumwwdeoxycholate with tauroiithocholate in the perfused rat liver completely prevented the decrease of bide flow aad the iuaease of oxygen uptake found with taurolithocholate only. Bile flow and bile salt secretion were inaeawd with the coiafusioa of both bile acidsas compared with the b&ion of tauroursodeoxychcdate only (+4.30& liver per 30 mia) with 16 and 32 ~molll tluroursodeoxycholate (+ 1.55 rug liver per 30 min with 80 and 16Oamobl). Morphological examination reveakd a 50% decrease of the number of necrctic cells in the periportal area. Taurounodeoxycholate did sot inhibit the uptake of t.mrolithocholate, but increased its transcelhdar passage and biotransformation. lotus. tauroursodeoxycbolate prevents taurolithocholate-induced cbotestasis and liver cell toxicity probably by an intracetlutar mecbar&m.
Ursadeoxycholate for the treahuent
(UDC) has recently been advocated of chronic liver diseases. in particular
in
cholestatic states such as primary biliary cirrhosis and primary sclerosing cholangitis (l-3). A decrease of several serum enzyme levels, of bilbuhin and, in one study (2). a histological improvement have been reported. However, the mechanism of this beneficial effect remains obscure. A protective effect of the taurine conjugate of UDC (TUDC) in bile duct ligated rats has been described carher (4). In addition, coinfusion of UDC led to a further increase of bile tlow when taurocholate was infused at wncentrations above its secretory maximum in the rat. With-
in a retrograde
way (9). It is kaown that the miecllc form-
ing bile salt taumcholate (TC) but not the aoamicelle forming k&-bile acid dehydrocholate (TDHC) protects against cholestasis when infused simultanously Evidence
has been presented,
in abstract
(10). form, that
TIJDC cat prevent liver damage to some exteot when coinfused with TLC iu the rat (11). In order to analyze further the mechauism of UDC liver pmtection we tested the ability of TDDC to prevent TLC iudued cholestasis in the isolated perfused rat liver analyzing the effect of two different mncentratioos of TUDC on uptake, metabolism and secretion
ofTLC,
and on bile flow changes.
out UDC this resulted in a decrease of bile flow (5). Taumlithocholate (TLC) causes cholestasis in the rat (6-S). An increased cholesterollphaspholipid ratio in a” membranes seem; to be a major mechanism. In addition, cellular necrosis in cells of zone 3 (perivenous), but not cholestasis when liven are perfused
nc causes extensive
Methods The isolated perfused rat liver was used as described (12). The liver was single pass perfus.d using albumin-
281 free Rrebs-Ringer bicarbonate (pH 7.4) gassed with carbogen at a flow rate of 4 ml per min per g liver. The p&usate cOntained lactate (2.1 ““W/l), pyrwete (0.3 mmov1), aceto-acetate (0.1 “m&l), B-hydroxybutyate (0.@8 mmolil), and glucox (5 mmolll). The flow was kept constarn and perfusion pressure WBS below 14 emH,O. Oxygen uptake was measured w&g two Clark-type slectrades in the influent and effluent nerfwate tubes and calculated 8s the difference of both. After 30 tin equilibredon, 0, uptake (2.6 f O.Z~moUg liver per tin), bile flow (1.04 5 0.08~Ugliverpermin), andbile salt secretion (6.3 + 0.8 ““to@ liver per min) were assessed. The” TLC (Signta, St. Louis, MO, U.S.A.) dissolved in propylene gWl(13) was added to the petfuwe to achieve a final co~“Pratio” of 8~atoUl. After 15 min. the concentration wes doubled to 16 pa&l. In other experimemr TUDC (Calbiwbem, San Diego, CA, U.S.A.) was added (perfusate macentratio” 16 /rmoM) and the” increased to 32 PmoVl efta 15 min. Alternatively, a concentration of 80 jmxdA wes wd initially end then increased to 16OpmoM. Finally. ccmbinatioas of TLC and TUDC were infused either at aratioof I:2 or 1:lOdependi”go” thti conantratio”ofTUDCused. Bii serttplcs were Follccted et S-mia interv& and bile flow was determined by gmvi”wy. Bile sample coneenhation e”d hydmxyfatio” pattern io bile sod perfusate were amfyzed using biolumjnescence assays (M-16). Using different assays - Sal-hydroxy bile salts (~-U-OHBS) and 7+hydroxy bile salts (7+OH-BS) - TLC end lWDCupt& a”d secretion could be separated. Further“tort, using l?a.OH-BSand 7-a.OH-BS arrays the amau”t of TLC metaboliizcd could be c&“lated, using the cquetions given in Ref. 17. The results of this assay technique were conuollcd by HPLC (18) and thin-layer chromatography. in some experiments revealing a good cotmlalio” (I?‘). Three perfusions were performed for each bile salt, concentration
and combination.
As expected, TLC cawed complete cholestasis during the perfusion period. 02 uptake imreawd by 6.7% (8 pm&l) and 8.1% (16,umobl). TJDC iocreased the bile flow slightly with 16 end 32 pmovl and to e larger extent with 80 and 160 ,wnot” in Ihe perf”sate. 0, upteke i”creased less than dtb TLC(Tebk 1). There was no sign&ant influence of TUDC on the uptake of TLC (Fig. 1). Bile flaw was not significantly different when TIJDC or TUDC + TLC was infused, regardless of the concentration used (Figs. 2 and 3). When a c”mulative calculatio” was used, the combination of TUDC end TLC induced more bile flow then TUDC alone. This was “tore pronounced Mb the lower dose of TUDC (Table 2). Accordingly, this combination TUDC alone.
bile salt secretion was higher with
(i250nmollgliverper3Omi”)
than with
1. ScH6LMERfcH
TLC was secreted unchanged or as biotransformation onducts LT-muricholate. 3-a-6-B-OH-BS and several oxo-hydroxyBS) almost completely, the metabolites representing between 35 and 40% of the total secretion (Fig. 4). The increase of oxygen consumption with TUDC + TLC was comparable to that of TUDC only and lower when comparedwith that induced by TLC (Table 1). Examination by light microscopyrevealed a small percentage (0.4 + 0.1%) of necrotic cells in the priportal zone of livers perfused with the combination. This number was lower than with TLC alone (1.28%) (9).
mscwlon
The data presented demonstrate that the taurine conjugate of the hydrophilic bile salt ursodeoxycholate (TUDC) prevents cholestasls and toxicity induced by the hydrophobic bile salt taurolithocholate (TLC). Several possible mechanismsare evident. First, TUDC could inhibit the uptake of TLC at the sinusoidal membrane, al-
et al.
though inhibition should probably be fx)t\compclitiuc (19). However, we did not find any uptake inhibition for TLC by TUDC. This is in accordancewith data showing that chalic acid protects against lithocbolate acid-induced chokstasis not via uptake inhibition but via an alteration of the TLC-cholesterol interaction (20). The tramcellular transport of TLC is increased by the coinfusion of TUDC. Fustbennore, the biotmmfmmation of TLC is increasedwhen comparedwith single infu. sion. Without TUDC, almost no bile salt was excretedafter lo-l.5 min of TLC infusion. Less than 20% of the total amouot infused was excreted dnring this period as bittransformation product or unchanged TLC (9). The~after, tiobansformationcouldnotbcasseMcdinaurmodel. TLC and its metabolites act as slightly cholereticbile sals in the winfusion system (Table 1). It might be wxtclt#kd that changes of intracellular compartmentatian or of the availability of TLC to proteins (21). which may act 8s bile acid-metabolizing enzymes (22) are responsible fox the protective effect. The effect is tbought to bc different fram that of cholate, since TUDC does riot produce 8 comparable cholesteml solubilization (20 and personal communication). TUDC does not only p~eveot tbs decrease of bile flow but, in addition, the increaseof oxygen uptake induced by TLC. Sbxe oxygen uptake is conelated with the toxicity of bile acids and not with uptake, transport or secretion (24). this can be taken as an indication of a reduction in toxicity, in accordancewith earlier data (11). This is forther supported by the finding cd a Rduced n-mber of necrotic cells in the winfusion experiItIe”&. It has been shown that UDC increasescholer&s when bile salts we infused above their secretory rate maximum (5). However, the secretory rate maximum is probably due to toxic effects of high concentrations of bile salts at
the canalicular level. In wntrast, the effect of TLC is different from that of tawocholate or taurachenodeoxycholate and takes place at much lower concentrations (25). Tbus, TLC probably affects the cantdicular membrane leadiig to cbolestasis and. in addition, has some intracellular to&by. s”awt that in zddition f~coonter-
acts similarly in the prevention of bile salt-induced changes as. the taurine conjugate (26), and an increase of toxic bile salts in cholestatic diseases has been found (27). this might explain&e beneficial effect of UDC in such diseases.
Ourdata
acting choles~atic effects (5) TUDC can prevent the cellular toxicity. In order to understand this phenomenon better. studier enel@g intracelbdar biotransformation are required. but these an diicult to perform. In summary, TUDC prevents TLC-induced &&stasis and liver cell toxicity by B mechanism related to intracellular metabolism, but not by ilptakc inhibition for TLC. Since it has been shown that the unconjugated bide salt
Exphfcd 1989:189:g-42. 14 srllMwr*h 1. Hinkky JE. McDcaatd LA, H&am,
AF, D&u-
Tlds study was supponed by NIH grant No. AM 161JO of the U.S. Public Health Service (K.M.). J.S. vas supported by the Tbyssen Fotmdation, Colo@e, F.R.G. ‘Ibis study has been presented as an abstract meeting 1988 in Leuven. Belgium.
at the EASL
prevents taurolithocholate-induced and toxicity in rat liver*
cholestasis
Ursodeoxycholate has been advocated for the treatment of cholestatic liver diseases. The coinfusion of taumwwdeoxycholate with tauroiithocholate in the perfused rat liver completely prevented the decrease of bide flow aad the iuaease of oxygen uptake found with taurolithocholate only. Bile flow and bile salt secretion were inaeawd with the coiafusioa of both bile acidsas compared with the b&ion of tauroursodeoxychcdate only (+4.30& liver per 30 mia) with 16 and 32 ~molll tluroursodeoxycholate (+ 1.55 rug liver per 30 min with 80 and 16Oamobl). Morphological examination reveakd a 50% decrease of the number of necrctic cells in the periportal area. Taurounodeoxycholate did sot inhibit the uptake of t.mrolithocholate, but increased its transcelhdar passage and biotransformation. lotus. tauroursodeoxycbolate prevents taurolithocholate-induced cbotestasis and liver cell toxicity probably by an intracetlutar mecbar&m.
Ursadeoxycholate for the treahuent
(UDC) has recently been advocated of chronic liver diseases. in particular
in
cholestatic states such as primary biliary cirrhosis and primary sclerosing cholangitis (l-3). A decrease of several serum enzyme levels, of bilbuhin and, in one study (2). a histological improvement have been reported. However, the mechanism of this beneficial effect remains obscure. A protective effect of the taurine conjugate of UDC (TUDC) in bile duct ligated rats has been described carher (4). In addition, coinfusion of UDC led to a further increase of bile tlow when taurocholate was infused at wncentrations above its secretory maximum in the rat. With-
in a retrograde
way (9). It is kaown that the miecllc form-
ing bile salt taumcholate (TC) but not the aoamicelle forming k&-bile acid dehydrocholate (TDHC) protects against cholestasis when infused simultanously Evidence
has been presented,
in abstract
(10). form, that
TIJDC cat prevent liver damage to some exteot when coinfused with TLC iu the rat (11). In order to analyze further the mechauism of UDC liver pmtection we tested the ability of TDDC to prevent TLC iudued cholestasis in the isolated perfused rat liver analyzing the effect of two different mncentratioos of TUDC on uptake, metabolism and secretion
ofTLC,
and on bile flow changes.
out UDC this resulted in a decrease of bile flow (5). Taumlithocholate (TLC) causes cholestasis in the rat (6-S). An increased cholesterollphaspholipid ratio in a” membranes seem; to be a major mechanism. In addition, cellular necrosis in cells of zone 3 (perivenous), but not cholestasis when liven are perfused
nc causes extensive
Methods The isolated perfused rat liver was used as described (12). The liver was single pass perfus.d using albumin-
281 free Rrebs-Ringer bicarbonate (pH 7.4) gassed with carbogen at a flow rate of 4 ml per min per g liver. The p&usate cOntained lactate (2.1 ““W/l), pyrwete (0.3 mmov1), aceto-acetate (0.1 “m&l), B-hydroxybutyate (0.@8 mmolil), and glucox (5 mmolll). The flow was kept constarn and perfusion pressure WBS below 14 emH,O. Oxygen uptake was measured w&g two Clark-type slectrades in the influent and effluent nerfwate tubes and calculated 8s the difference of both. After 30 tin equilibredon, 0, uptake (2.6 f O.Z~moUg liver per tin), bile flow (1.04 5 0.08~Ugliverpermin), andbile salt secretion (6.3 + 0.8 ““to@ liver per min) were assessed. The” TLC (Signta, St. Louis, MO, U.S.A.) dissolved in propylene gWl(13) was added to the petfuwe to achieve a final co~“Pratio” of 8~atoUl. After 15 min. the concentration wes doubled to 16 pa&l. In other experimemr TUDC (Calbiwbem, San Diego, CA, U.S.A.) was added (perfusate macentratio” 16 /rmoM) and the” increased to 32 PmoVl efta 15 min. Alternatively, a concentration of 80 jmxdA wes wd initially end then increased to 16OpmoM. Finally. ccmbinatioas of TLC and TUDC were infused either at aratioof I:2 or 1:lOdependi”go” thti conantratio”ofTUDCused. Bii serttplcs were Follccted et S-mia interv& and bile flow was determined by gmvi”wy. Bile sample coneenhation e”d hydmxyfatio” pattern io bile sod perfusate were amfyzed using biolumjnescence assays (M-16). Using different assays - Sal-hydroxy bile salts (~-U-OHBS) and 7+hydroxy bile salts (7+OH-BS) - TLC end lWDCupt& a”d secretion could be separated. Further“tort, using l?a.OH-BSand 7-a.OH-BS arrays the amau”t of TLC metaboliizcd could be c&“lated, using the cquetions given in Ref. 17. The results of this assay technique were conuollcd by HPLC (18) and thin-layer chromatography. in some experiments revealing a good cotmlalio” (I?‘). Three perfusions were performed for each bile salt, concentration
and combination.
As expected, TLC cawed complete cholestasis during the perfusion period. 02 uptake imreawd by 6.7% (8 pm&l) and 8.1% (16,umobl). TJDC iocreased the bile flow slightly with 16 end 32 pmovl and to e larger extent with 80 and 160 ,wnot” in Ihe perf”sate. 0, upteke i”creased less than dtb TLC(Tebk 1). There was no sign&ant influence of TUDC on the uptake of TLC (Fig. 1). Bile flaw was not significantly different when TIJDC or TUDC + TLC was infused, regardless of the concentration used (Figs. 2 and 3). When a c”mulative calculatio” was used, the combination of TUDC end TLC induced more bile flow then TUDC alone. This was “tore pronounced Mb the lower dose of TUDC (Table 2). Accordingly, this combination TUDC alone.
bile salt secretion was higher with
(i250nmollgliverper3Omi”)
than with
1. ScH6LMERfcH
TLC was secreted unchanged or as biotransformation onducts LT-muricholate. 3-a-6-B-OH-BS and several oxo-hydroxyBS) almost completely, the metabolites representing between 35 and 40% of the total secretion (Fig. 4). The increase of oxygen consumption with TUDC + TLC was comparable to that of TUDC only and lower when comparedwith that induced by TLC (Table 1). Examination by light microscopyrevealed a small percentage (0.4 + 0.1%) of necrotic cells in the priportal zone of livers perfused with the combination. This number was lower than with TLC alone (1.28%) (9).
mscwlon
The data presented demonstrate that the taurine conjugate of the hydrophilic bile salt ursodeoxycholate (TUDC) prevents cholestasls and toxicity induced by the hydrophobic bile salt taurolithocholate (TLC). Several possible mechanismsare evident. First, TUDC could inhibit the uptake of TLC at the sinusoidal membrane, al-
et al.
though inhibition should probably be fx)t\compclitiuc (19). However, we did not find any uptake inhibition for TLC by TUDC. This is in accordancewith data showing that chalic acid protects against lithocbolate acid-induced chokstasis not via uptake inhibition but via an alteration of the TLC-cholesterol interaction (20). The tramcellular transport of TLC is increased by the coinfusion of TUDC. Fustbennore, the biotmmfmmation of TLC is increasedwhen comparedwith single infu. sion. Without TUDC, almost no bile salt was excretedafter lo-l.5 min of TLC infusion. Less than 20% of the total amouot infused was excreted dnring this period as bittransformation product or unchanged TLC (9). The~after, tiobansformationcouldnotbcasseMcdinaurmodel. TLC and its metabolites act as slightly cholereticbile sals in the winfusion system (Table 1). It might be wxtclt#kd that changes of intracellular compartmentatian or of the availability of TLC to proteins (21). which may act 8s bile acid-metabolizing enzymes (22) are responsible fox the protective effect. The effect is tbought to bc different fram that of cholate, since TUDC does riot produce 8 comparable cholesteml solubilization (20 and personal communication). TUDC does not only p~eveot tbs decrease of bile flow but, in addition, the increaseof oxygen uptake induced by TLC. Sbxe oxygen uptake is conelated with the toxicity of bile acids and not with uptake, transport or secretion (24). this can be taken as an indication of a reduction in toxicity, in accordancewith earlier data (11). This is forther supported by the finding cd a Rduced n-mber of necrotic cells in the winfusion experiItIe”&. It has been shown that UDC increasescholer&s when bile salts we infused above their secretory rate maximum (5). However, the secretory rate maximum is probably due to toxic effects of high concentrations of bile salts at
the canalicular level. In wntrast, the effect of TLC is different from that of tawocholate or taurachenodeoxycholate and takes place at much lower concentrations (25). Tbus, TLC probably affects the cantdicular membrane leadiig to cbolestasis and. in addition, has some intracellular to&by. s”awt that in zddition f~coonter-
acts similarly in the prevention of bile salt-induced changes as. the taurine conjugate (26), and an increase of toxic bile salts in cholestatic diseases has been found (27). this might explain&e beneficial effect of UDC in such diseases.
Ourdata
acting choles~atic effects (5) TUDC can prevent the cellular toxicity. In order to understand this phenomenon better. studier enel@g intracelbdar biotransformation are required. but these an diicult to perform. In summary, TUDC prevents TLC-induced &&stasis and liver cell toxicity by B mechanism related to intracellular metabolism, but not by ilptakc inhibition for TLC. Since it has been shown that the unconjugated bide salt
Exphfcd 1989:189:g-42. 14 srllMwr*h 1. Hinkky JE. McDcaatd LA, H&am,
AF, D&u-
Tlds study was supponed by NIH grant No. AM 161JO of the U.S. Public Health Service (K.M.). J.S. vas supported by the Tbyssen Fotmdation, Colo@e, F.R.G. ‘Ibis study has been presented as an abstract meeting 1988 in Leuven. Belgium.
at the EASL