TOXICOLOGICALSCIENCES108(2),225–246(2009) doi:10.1093/toxsci/kfn268 AdvanceAccesspublicationJanuary8,2009 REVIEW Bile Acid Sulfation: A Pathway of Bile Acid Elimination and Detoxification Yazen Alnouti1 DepartmentofPharmaceuticalSciences,CollegeofPharmacy,UniversityofNebraskaMedicalCenter,Omaha,Nebraska68198 D o w n ReceivedOctober14,2008;acceptedDecember24,2008 lo a d e Sulfotransferase-2A1 catalyzes the formation of bile acid- minahyesaeltrhveaansdadniseeffiasceie.ntmechanismtomaintainBAhomeostasis d from sulfates (BA-sulfates). Sulfation of BAs increases their solubility, DespitetheimportanceofsulfationinBAresearch,thereare h decreasestheirintestinalabsorption,andenhancestheirfecaland ttp no recent reviews of this topic (Stiehl, 1974b), whereas s urinary excretion. BA-sulfates are also less toxic than their hundreds of reviews on other aspects of BA research are ://a unsulfated counterparts. Therefore, sulfation is an important ca available. Therefore, this review focuses on several aspects d detoxification pathway of BAs. Major species differences in BA e m sulfationexist.Inhumans,onlyasmallproportionofBAsinbile related to BA sulfation including BA-sulfate formation, ic and serum are sulfated, whereas more than 70% of BAs in urine elimination, toxicity, and regulation. Because there are major .ou p are sulfated, indicating their efficient elimination in urine. The differences in the BA physiologic and pathologic effects .c o formation of BA-sulfates increases during cholestatic diseases. amongvariousspecies,thisreviewfocusesonhumandata,and m Therefore, sulfation may play an important role in maintaining occasionally presents animal data to compare with or support /to x BA homeostasis under pathologic conditions. Farnesoid X re- human data. Because this topic has not been reviewed for sc ceptor,pregnaneXreceptor,constitutiveandrostanereceptor,and i/a vitamin D receptor are potential nuclear receptors that may be aholwonogldtimthee,yaanreo)viesrvpireewsenotfeda,llindaatnaaatvteamilapbtleto(lriengkarbdolethssooldf rticle involvedintheregulationofBAsulfation.Thisreviewhighlights -a andcurrentdata.Finally,asindicatedinthetext,BAsynthesis b current knowledge about the enzymes and transporters involved and enterohepatic recirculation have been the subject for stra in the formation and elimination of BA-sulfates, the effect of c sulfationonthepharmacologicandtoxicologicpropertiesofBAs, several reviews. However, relevant information from these t/10 theroleofBAsulfationincholestaticdiseases,andtheregulation reviewswillstillbediscussedinsomedetailtodemonstratethe 8/2 of BA sulfation. marked influence of sulfation on BA disposition. /2 2 5 Key Words: bileacids; sulfate; sulfation;sulfonate; sulfonation; /1 6 sulfotransferase; SULT; nuclear receptors; gender difference; 6 BASYNTHESIS 4 regulation; homeostasis;cholestasis. 3 8 3 Cholesterol is the building block of BA synthesis. Detailed by g description of the enzymes involved in BA synthesis can be u e Bile acids (BAs) are synthesized in the liver, from the foundinarecentreview(Russell,2003).Themajorpathwayof st o oxidationofcholesterol, andsecretedintoduodenum.BAs are BAsynthesisiscalledtheneutralorclassicalpathwayanditis n 2 conserved through efficient enterohepatic recycling. BAs have initiatedwithcholesterolhydroxylationatthe7apositionbythe 7 M many physiologic functions including the regulation of the microsomalCYP7A1enzyme,whichisexclusivelyexpressedin arc expression of genes involved in cholesterol, glucose, and their theliver (Chiang, 1998). Next, is the classical oxido-reduction h 2 own homeostasis. However, BAs also have several pathologic stepinsteroidogenesis,whichincludestheoxidationofthe3b- 01 9 effects including carcinogenicity and liver toxicity. Therefore, OH and isomerization of the C5-C6 double bond by the maintenance of BA homeostasis is essential to achieve their microsomal C27-3b-hydroxysteroid dehydrogenase (C27-3b- physiologic functions and avoid their toxic effects. HSD)(Schwarzetal.,2000).Theresultingintermediateiseither, Sulfationisanimportantmetabolicpathwaytodetoxifyand hydroxylatedatthe12apositionbythemicrosomalCYP8B1or, eliminateBAs.BA-sulfatesaremorewatersolubleandtherefore passedondirectlytothenextstep(Gafvelsetal.,1999).The12a- aremorereadilyexcretedinfecesandurine.Furthermore,BA- hydroxylatedintermediatesandthosethatescaped12ahydrox- sulfatesarelesstoxicthanunsulfatedBAs.Therefore,sulfation ylationhavetheirC3oxoandtheC4-C5doublebondreducedto yield 3a-OH intermediates by the cytosolic oxosteroid 5b- reductase and 3a-HSD (members of the aldo-keto reducatse 1Forcorrespondenceviafax:Fax:(402)559-9543.E-mail:[email protected]. family)(Penningetal.,2004).12ahydroxylationwillultimately (cid:1)TheAuthor2009.PublishedbyOxfordUniversityPressonbehalfoftheSocietyofToxicology.Allrightsreserved. Forpermissions,pleaseemail:[email protected] 226 ALNOUTI produce cholic acid (CA), whereas intermediates that were not completeionizationofBAsatphysiologicalpH,whichmarkedly hydroxylated will ultimately produce chenodeoxycholic acid increases their solubility and therefore, decreases membrane (CDCA).CAandCDCAaretheprimaryBAsinhumans. permeability(Guetal.,1992;HofmannandMysels,1992). Other primaryBAs aresynthesizedinotherspeciessuchas, AlternativepathwaysforBAsynthesis,whichdonotrequire hyocholic acid in pigs (Lundell et al., 2001), a- and b- theinitiationbyCYP7A1,arealsoknown(AxelsonandSjovall, muricholic acid (MCA) in rodents, and ursodeoxycholic acid 1990).Thesepathwaysareinitiatedviathehydroxylationofthe (UDCA)inbears(Russell,2003).Figure1showsthechemical cholesterol side chain at the C24, C25, or C27 positions by structure of the most common BAs in mammals. various enzymes such as, CYP27A1 (Lund et al., 1993) and The next step in BA synthesis is the hydroxylation and CYP46A1(Lundetal.,1999).Theresultingoxysterolsarethen oxidation to a carboxylic acid at the C27 position by the hydroxylatedatthe7apositionbyCYP7B1(Roseetal.,1997; mitochondrial CYP27A1 enzyme (Pikuleva et al., 1998), Schwarzetal.,1998)andCYP39A1(Li-Hawkinsetal.,2000) D o followed by ligation to coenzyme A by the bile acid co- instead of CYP8B1. In contrast to the classical pathway, these w n enzyme-A synthetase (BAS) (Mihalik et al., 2002). The side alternativepathwaysproducepredominantlyCDCA(Vlahcevic lo a chainsoftheseC27intermediatesarethenshortenedtoC24BAs etal.,1999).AlternativeBApathwaysseemmoreimportantin de d byb-oxidationintheperoxisomes(NorlinandWikvall,2007). conditions associated with deficiency in CYP7A1 activity fro ThefinalstepinBAsynthesisistheamidationoftheBA-CoA (Axelsonetal.,1989). m h with an amino acid, usually glycine (G) or taurine (T), by the ttp s BA-CoA:amino acid N-acyltransferase (BAT) (Solaas et al., ://a 2000). BAs are almost exclusively synthesized and excreted ENTEROHEPATIC RECYCLING OFBAs ca d from the liver in the amidated form. G-amidates are predomi- e m nant in humans (Falany et al., 1994), whereas T-amidates are BAs synthesized in the liver are secreted into bile, which ic .o predominant in rodents (Falany et al., 1997). Amidation in- flowsthroughthebile duct totheintestine. BAs are efficiently u p creasestheacidityofunconjugatedBAs,wherepKAisreduced absorbedfromtheintestine,returnedtotheliver,andresecreted .c o m fromabout5.5fortheunconjugatedBAsto4.5and1.5forthose into bile. Thiscycle iscalledthe enterohepatic recirculation of /to with G- and T-amidation, respectively (Falany et al., 1997; BAs, and was recently reviewed (Hofmann and Hagey, 2008). x s c Hofmann and Hagey, 2008; Russell, 2003). This results in BAsareexcretedfromtheliverintobileviaeffluxtransporters i/a locatedinthecanalicularmembrane.AmidatedBAsareexcreted rtic le intobileviathecanalicularbilesaltexportpump(BSEP)(Byrne -a b et al., 2002; Noe et al., 2002). In contrast, unconjugated BAs s generallyhaveloweraffinity(Gerloffetal.,1998)ornoaffinity trac (Noeetal.,2002;Zelceretal.,2003)forBSEP.Inmanyspecies t/1 0 8 including humans, most of the secreted bile is stored in gall- /2 bladder,which,undertheinfluenceofcholycystokininsecretion /22 5 after meal ingestion, contracts to empty its contents into the /1 6 duodenum. Other species, such as the rat, do not have a gall- 64 3 bladder and therefore, most of its secreted bile is stored in the 8 3 smallintestine(HofmannandHagey,2008). by g In the small intestine, most amidated BAs are actively u e absorbedintheileum(Aldinietal.,1996;Kuipersetal.,1988) st o by the apical Naþ-dependent bile salt transporter (ASBT) n 2 located on the apical side of enterocytes (Wong et al., 1995). 7 M Organicanion-transportingpolypeptides(OATPs),whichhave a rc aloweraffinityforBAsthanASBT(Waltersetal.,2000),may h 2 also contribute to BA transport across the apical membrane 01 9 throughout the intestine. Both transporters have higher affinity for amidated rather than unconjugated BAs (Craddock et al., 1998; Walters et al., 2000). Because unconjugated BAs are mostly non-ionized and because they have low affinity for ASBT, passive rather than active absorption is the primary routeofabsorptionthroughouttheintestinaltract(Aldinietal., 1996; Mekhjian et al., 1979; Schiff et al., 1972; Takikawa et al., 1997b). In contrast, because of their high affinity for ASBT, and because they are mostly ionized at intestinal pH, FIG.1. ThechemicalstructureofthemostcommonBAsinmammals. active transport represents the major route of absorption of BILEACIDSULFATION 227 amidated BAs (Schiff et al., 1972). Vectorial BA transport mediated by several transporters including OSTa, and b fromtheintestinallumentobloodisthenaccomplishedbythe (Ballatori et al., 2005), and multidrug resistance-associated organic solute transporters (OST-a and b) located on the proteins (MRP1, MRP3, MRP4, and MRP6) (Trauner et al., basolateral side of enterocytes (Ballatori et al., 2005; Dawson 2005).Undernormalconditions,mostBAsarecontainedwithin et al., 2005; Rao et al., 2008). Partial de-amidation, especially the enterohepatic system, with minimal spill over into blood, oftheG-amidates,takesplacebythebacteriainthedistalparts and negligible urinary excretion (Baumgartner et al., 1995). of the small intestine, and the liberated unconjugated BAs are Undercholestaticconditions,suchasbileductobstruction,BA passively absorbed (Hepner et al., 1972a,b, 1973). efflux through the sinusoidal membrane, facilitated by the Unabsorbed BAs are passed along from the small to large upregulationofthesinusoidaleffluxtransporters,becomesmore intestine. In the large intestine, BAs undergo bacterial trans- significant(Baumgartneretal.,1995;TraunerandBoyer,2003; formation, including de-amidation and dehydroxylation. Pri- Zollneretal.,2007). Therefore,urinaryexcretion becomesthe D o mary BAs are dehydroxylated at the 7a position by colonic primary route of BA elimination. In the kidney, unbound w n bacteria to produce secondary BAs (Lundeen and Savage, fractions of BAs are filtered through the glomerulus and lo a 1990).Deoxycholicacid(DCA)andlithocholicacid(LCA)are reabsorbedefficientlythroughtheapicalmembraneofproximal de d secondary BAs producedfrom thedehydroxylationofCA and tubules in a similar manner to their absorption in the intestine fro CDCA, respectively (Hofmann and Hagey, 2008). Dehydrox- (Barnes et al., 1977), presumably via ASBT (Christie et al., m h ylation is performed by strict anaerobic bacteria, whereas de- 1996),andOATPs(TraunerandBoyer,2003).Intheopposite ttp s amidationisperformedbyboth aerobicandanaerobicbacteria direction,activetubularsecretionintourinemayalsotakeplace, ://a (Yesair andHimmelfarb, 1970). Therefore, de-amidationtakes presumablyvia(MRP2)andorganicaniontransporters(OATs) c a d placeinboththesmallandlargeintestines,whereasdehydrox- (St-Pierreetal.,2001). e m ylationonlyoccursinthelargeintestine.Usingisolatedintestinal ic .o bacterialstrains,itwasdemonstratedthatde-amidationisapre- u p requirement for BA dehydroxylation (Gustafsson et al., 1968; BA SULFATIONINHUMANS .c o m Midtvedt, 1974). However, limited dehydroxylation of BAs- Sulfation is a phase II conjugation reaction that transfers /to amidatesstilloccursinvivo(Hepneretal.,1972a).ASBTisnot x aTbhuenredfaonrtel,ymeixnpimreaslseadctiivneathbesocrpotlioonn(oCfrtahdedionctakcteBtAa-l.a,m1i9d9at8e)s. paamhsouinslfpeoh,nooaartdeceganrroobsuoipnxey(Sli5Oc#3(cid:1)-ap)chifdorosgpmrhootuhspeulufoanftievae(rsPsuaAblsPstSural)tf,eo.tnoaTthaeedhoryendsorurol,txi3yn#lg-, sci/article takesplaceinthelargeintestine(Kuipersetal.,1988;Mekhjian sulfate-conjugates carry a permanent negative charge (pKa of -ab etal.,1979),whereasunconjugatedprimaryandsecondaryBAs s arepassivelyabsorbed.BAsthatarenotabsorbedfromthesmall sulfate moiety < 1), and therefore, are very water soluble. trac andlargeintestineundergofurtherbacterialmetabolism,suchas ‘‘Sulfonation’’ is actually the correct term to describe this t/1 metabolic pathway; however, the less proper term ‘‘sulfation’’ 08 reductionandepimerizationoftheirOHgroupsfromthea-tothe /2 b- conformation, before excretion in feces (Franklund et al., waschosenbecauseithasbeenhistoricallypopular,especially /22 in BA research. Sulfation is catalyzed by a group of enzymes 5 1990; Macdonald et al., 1983). Ninety-five percent of BAs /1 called sulfotransferases (SULTs) (Glatt, 2000). BAs have 6 excretedinbilearereabsorbedthroughouttheintestinaltractand 64 severalOHgroupsthatcanbetargetsforsulfation;however,as 3 less than 5% are excreted in feces, mostly as secondary 8 unconjugated BAs (Hamilton et al., 2007; Hofmann, 1999a; will be discussed later, mono-sulfation at the 3-OH position is 3 by predominant in humans. Figure 2 depicts the enzymatic g Beheretal.,1984;Mosbach,1972;MosbachandSalen,1974; u formation of BA-3 sulfates. e Owen et al., 1984, 1987a,b; Pacini et al., 1987; Radominska The formation of BA-sulfated metabolites was recognized st o etal.,1993;vanGorkometal.,2002). n early as a mechanism of BA elimination in humans (Palmer, 2 AbsorbedBAsarecarriedintheportalveinandextractedby 1967). LCA sulfation was the first to be discovered and it was 7 M the liver via the Naþ-taurocholate cotrasporting polypeptide a estimated that 40–75% ofLCA inhuman bile ispresent inthe rc (NTCP),membersoftheOATPfamily(HagenbuchandMeier, h sulfated form (Palmer and Bolt, 1971). After i.v. or oral ad- 2 1994;Hagenbuchetal.,1991;Jacqueminetal.,1994),fattyacid 01 ministration to healthy humans, LCA and its amidates were 9 transport protein (Doege et al., 2006), and/or via passive shown to be sulfated and primarily excreted in bile in the diffusion (Ko et al., 1994). NTCP has a higher affinity for the sulfated-amidated (divalent) form (Allan et al., 1976; Cowen amidatedcomparedwiththeunconjugatedBAs(Schroederetal., 1998). In hepatocytes, most BAs are amidated, but other metabolic pathways take place such as, hydroxylation and sulfate or glucuronide conjugation (Radominska et al., 1993). ThereabsorbedandnewlysynthesizedBAsarethenexcretedin biletocompletetheenterohepaticcycle. Efflux of BAs out of hepatocytes into the systemic blood through the sinusoidal membrane also takes place and is FIG.2. TheformationofBA-3-monosulfatescatalyzedbySULT2A1. 228 ALNOUTI et al., 1975a,c). However, sulfation of CDCA and CA Takingthese data collectively, itcan be concludedthat BA- administered to cirrhotic patients was much less than LCA, sulfates are exclusively present in the mono-sulfate form in andtherateofCAsulfationestimatedtobehalfthatofCDCA humans and rodents. Conflicting data have been reported (Stiehl, 1974a; Stiehl et al., 1975). All endogenous BAs may regarding the position of sulfation, but sulfation at the 3-OH be detected in the sulfated form, but in various proportions. and 7-OH is likely to be predominant in humans and rodents, SimilartounsulfatedBAs,greaterthan90%ofBA-sulfatesare respectively. also amidated (divalent), with G-amidates being predominant in humans (Goto et al., 2007; Stiehl et al., 1985). EFFECT OFSULFATIONON THEENTEROHEPATIC Structure of BA-Sulfates in Humans RECIRCULATIONOFBAs D Conflicting data regarding the structural elucidation of BA- BA-sulfates undergo limited enterohepatic recirculation ow sulfateshavebeenreported.Itwasreportedthatinhumanurine, n because of their limited absorption through the intestine and lo 77%ofBA-sulfatesaremono-sulfates,21%aredi-sulfates,and a their limited extraction by the liver. In addition, transporters d e 2%aretri-sulfates(Stiehl,1974a;Stiehletal.,1982).Inaddition, d equal proportions of mono- and poly-sulfated BAs in human and bacterial transformation pathways involved in BA-sulfate fro absorption, metabolism, and excretion are different than those m uaBnrAidn-etsruiw-lsfeuartleefasdtee(tdBecBatreAthdso(wlRoeamreeedwaslcshoeteftoaaul.ln,.,d11t99o88c21o))n..sIBtniothuthutemBleaAsns-p3thl-aasnamn5da%,-d7oi--f foonrIttuhwnesaeusnlftraeetrceoodhgeBnpiAaztesid.cFrtehigcauytrcBeliAn3g-islouluflfsaBttrAeastse.sarteheexecfrfeetcetdoifnsublifleativoina https://ac sulfatesweredetectedattheratioof2:1(Raedschetal.,1981). adifferentpathwaythanunsulfatedBAs.Thebiliaryexcretion ade Other reports, however,only detectedBA-mono-sulfates. In m of BA-glucuronide and -sulfate conjugates is reduced in the ic human urine, only mono-sulfates at the 3-OH position and no natural jaundice Groningen yellow (GY) rat and the Eisai .ou poly-sulfatesweredetected(Almeetal.,1977;Hedenborgand p hyperbilirubinemic (EHB) rat (naturally Mrp2 deficient rats), .c Norman, 1984). Furthermore, it was shown that the small whereasthebiliaryexcretionofunconjugatedBAsintheseratsis om di- and tri-sulfate fractions were actually artifacts due to notaffected(Kuipersetal.,1988,1989;Takikawaetal.,1991a). /tox Fcsaoumnrttphalemeripneravetiipodanernatcwieointhth(aAtthlmeoenBlyeAt-Ba3lA-.,m-1mo9no7on7-o;s-usBlufaalrftnaeteesfsreatacttaiolt.hn,e1d93u7-r9OibnH)g. IEthnHeciBroanrbtaritalislttiyvteotorswtrwialdne-srtpeyopuretnuarabntlsseu,ltmfoaettemrdabnBrsaApnoserwtveBassAicn-loseutslafpfafrteeecpsta,erdwedh(Aefrrkoeiamtas sci/article position are formed was provided from in vitro studies. Using et al., 2001). In addition, dibromosulphthalein (DBSP) (Mrp2 -abs BA-sulfating protein fractions, purified from human livers, tra c only BA-3-mono-sulfates were formed. Furthermore, incuba- t/1 tion with BA-mono-sulfates and -di-sulfates did not result in 08 /2 the formation of BA-poly-sulfates (Loof and Hjerten, 1980) /2 2 5 /1 6 Structure of BA-Sulfates in Animals 6 4 3 The position at which BAs are sulfated was also studied in 8 3 otherspecies.Onlymono-sulfatemetaboliteswereformedafter by g incubating CDCA with rat and hamster liver homogenates. u e Sulfation occurred at both the 3-OH and 7-OH positions st o althoughtheformationratesofBA-3-sulfateswereatleastfive n 2 times higher than that of BA-7-sulfates. Di-sulfate formation 7 M was negligible, and CDCA-3- and -7-mono-sulfates were not a rc substratesfor poly-sulfate formation(Kirkpatricketal., 1980). h 2 It was also shown that, in rat hepatocytes, DCA and CDCA FIG.3. TheeffectofsulfationonBAsEnterohepaticRecycling:Sulfation 01 9 were only metabolized to 3-mono-sulfates (Lambiotte and ofBAstakesplaceinthelivertoproduceBA-sulfates[taurine-BA-(T-BA-S), Thierry, 1980). glycine-BA- (G-BA-S), and nonamidated BA-sulfates (BA-S)]. In the small intestinenodeconjugationofthesulfatemoietytakesplaceandlimitedamount Using a rat kidney homogenate, LCA and CDCA were ofBA-sulfatesareabsorbedintact.AbsorbedBA-sulfatesareextractedintact sulfated at the 3-OH and 7-OH positions, respectively (Chen bytheliverlessefficientlythanunsulfatedBAs.BA-sulfatesinthesystemic et al., 1978a; Summerfield et al., 1976). In hamsters, blood are efficiently excreted in urine by the kidney. The majority of BA- incubation ofCDCA with varioustissue homogenates resulted sulfatesarenotabsorbedinthesmallintestineandarepassedintactintothe in the formation of mono-sulfates at the 7-OH position only largeintestine.BA-sulfatesaredesulfated,thesulfatemoietyisabsorbedand largely excreted in urine. BA amidates are mostly deamidated. The released withoutanydi-sulfates(Barnesetal.,1979b).Mono-sulfatesat sulfate (S), Glycine (G), and taurine (T) are mostly absorbed. The released the 7-OH position were the only BA-sulfates detected in mice unconjugated BAs are biotransformed to secondary BAs and reabsorbed or feces (Eyssen et al., 1976). excretedinthefecesassecondaryBAs. BILEACIDSULFATION 229 substrate) and BA-sulfates inhibit the biliary excretion of each uptakeofBA-sulfatesacrossthesinusoidalsideofhepatocytes other, whereas no interaction in the biliary excretion of is not well characterized. Sulfated and unsulfated BAs unsulfatedBAsandDBSPcanbedemonstrated(EngandJavitt, competitivelyinhibittheuptakeofeachotherintohepatocytes, 1983;Uegakietal.,1999).Usingcellsdoubletransfectedwith and therefore might in part share the same transport system Mrp2andBsep,itwasthenconfirmedthatMrp2isrequiredto across the sinusoidal membrane (Bartholomew and Billing, transport divalent BAs (sulfated- or glucuronidated-amidated 1983). However, hepatocytes have lower uptake capacity BAs), whereas Bsep is required to transport monovalent BAs toward sulfated BAs compared with unsulfated BAs (Bartho- (amidated BAs) (Akita et al., 2001; Stieger et al., 2000). lomew and Billing, 1983; Gartner et al., 1990). It was shown However,itwasshownthatdivalentBAshaveequalaffinityfor that BA-sulfates are substrates for the Oatp1 and Oatp2 human BSEP and MRP2 (Hayashi et al., 2005). Therefore, in localized on the sinusoidal side of hepatocytes in rats (Meng contrasttotherat,BA-sulfatesinhumansmightbesecretedinto et al., 2002; Reichel et al., 1999). The vectorial transport of D o bileviabothBSEPandMRP2. sulfated BAs from the sinusoidal to the canalicular side was w n Sulfation of BAs markedly decreases their absorption rate, demonstrated using OATP2/Mrp2 double transfected cells lo a preventstheirpassiveabsorptionfromthesmallintestineandthe (Sasakietal.,2002).NTCPmayalsocontributetoBA-sulfates de d colon,andrestrictstheirabsorptionsitetotheileum(DeWittand uptakebecausenor-CA-sulfatewasshowntobeasubstratefor fro Lack, 1980; Low-Beer et al., 1969; Walker et al., 1986). In NTCP (Schwab et al., 1997). Along with other BAs, newly m h addition, BA-sulfates are poor substrates/inhibitors for ASBT synthesized and recirculating BA-sulfates are excreted in bile ttp s (Craddocketal.,1998).Sulfationatthe7-OHpositionreduces to complete the enterohepatic cycle. ://a intestinal absorption more than 3-OH sulfation (De Witt and Similar to unsulfated BA, the major route of BA-sulfate c a d Lack, 1980; Rodrigues et al., 1995). Therefore, sulfation eliminationisbiliaryexcretion.Lessthan5%ofanLCA-sulfate e m decreases BA absorption and enhances their fecal excretion in doseadministeredtohealthyhumansisexcretedinurine(Allan ic .o therat(Palmer,1971),guineapig(Low-Beeretal.,1969),andin et al., 1976; Cowen et al., 1975b). However, in cholestatic u p man (Cowen et al., 1975b). However one report demonstrated conditions,BAsincludingBA-sulfatesaretransportedoutofthe .c o m that intestinal absorption of BA-sulfates was as complete as liver into systemic blood across the sinusoidal membrane. /to unsulfatedBAsinrats(Kuipersetal.,1986). SulfatedBAsarebettersubstratesthanunsulfatedBAsforMrp3 x s c ThesmallportionofBA-sulfatesthatisabsorbedfromsmall (Hirohashi et al., 2000) and Mrp4 (Zelcer et al., 2003), which i/a intestine is absorbed intact, and is excreted in bile without expressions are upregulated during cholestasis (Denk et al., rtic le desulfation during intestinal absorption, hepatic extraction, or 2004;Mennoneetal.,2006).UsingMrp4andMrp3nullmice,it -a b biliary excretion (Cowen et al., 1975a,b). However, by double was demonstrated that Mrp4 rather than Mrp3 may play an s labelingthesteroidandthesulfatemoieties,andbyexamining important role in the efflux of BA-sulfates into systemic trac the fecal content, it was demonstrated that BA-sulfates are circulation across the sinusoidal side of hepatocytes (Belinsky t/1 0 8 progressively desulfated before fecal excretion (Cowen et al., etal.,2005;Mennoneetal.,2006;Sakamotoetal.,2006;Zelcer /2 1975b; Palmer, 1971).The released sulfate moietywas mostly etal.,2006).OthertransportersknowntocontributetotheBA /22 5 recovered in urine. In addition, both rat and human feces have spill over into systemic blood during cholestasis such as Osta, /1 6 high desulfation activity toward BA-3-sulfates. However, BAs andb,Mrp1,andMrp6mayalsocontributetotheeffluxofBA- 64 3 sulfatedatthe7and/or12positionsareresistanttodesulfation, sulfates(Ballatorietal.,2005;Trauneretal.,2005) 8 3 andtherefore,ifexist,areexcretedintactinfeces(Pacinietal., With complete cholestasis, urinary excretion becomes the by g 1987; Huijghebaert et al., 1984). In general, BA-sulfates are primary route of BA and BA-sulfate excretion. Urinary u e resistant to any microbial transformations until the sulfate clearance of BA-sulfates in cholestasis, however, is 10- to st o moietyisdeconjugated(Pacinietal.,1987;Huijghebaertetal., 100-foldhigherthanthatoftheunsulfatedBAs(Corbettetal., n 2 1984).Therefore,mostBA-sulfatesaredesulfatedfirst,andthe 1981; Makino et al., 1975; Stiehl, 1974a; Stiehl et al., 1975). 7 M liberated BAs undergo bacterial transformation before being Therefore, serum levels of BA-sulfates remain relatively low, a rc absorbed in intestine or excreted in feces. BA-sulfates are whereas urinary levels become very high. The high urinary h 2 desulfatedwhenincubatedwiththecontentofthelarge,butnot clearance of BA-sulfates may be due to low extraction by the 01 9 the small, intestine indicating that, in contrast to deamidation, liver, inhibition of renal reabsorption by the unsulfated BAs desulfation does not take place before the large bowel (Barnes et al., 1977, 1979a; Summerfield et al., 1977), BA (Huijghebaert et al., 1984). Therefore, the major contribution sulfationbythekidney(Summerfieldetal.,1977),lowaffinity of sulfation is to bypass absorption in the small intestine by toASBT(St-Pierreetal.,2001),and/oractivetubularsecretion avoiding bacterial transformation to absorbable BA species of BA-sulfates (Corbett et al., 1981). (unconjugated and amidated BAs). BA-sulfates are then In summary, sulfation influences the enterohepatic recircu- deconjugated in the large intestine, where BA absorption is lation of BAs through decreasing their intestinal absorption, much less than that in the small intestine. hepaticextraction,andrenalreabsorption.Thismarkedlylimits Similar to unsulfated BAs, BA-sulfates absorbed in the theenterohepaticrecirculationofBAsandshiftsittowardfecal intestine are carried through the portal vein to the liver. The and renal excretion. 230 ALNOUTI IDENTIFICATIONAND CHARACTERIZATIONOF Inhumans,SULT2A1mRNAisabundantlyexpressedinthe BA-SULTs liver,intestine,andadrenalgland(Dooleyetal.,2000;Falany, 1997; Her et al., 1996; Javitt et al., 2001; Otterness et al., InhumansBA-sulfotransferaseactivitywasmainlydetected 1995). Some of these studies demonstrated that Sult2a1 was inliver(Chenetal.,1978b;Kirkpatrick etal.,1988;Loofand not expressed in kidney, colon, and stomach (Dooley et al., Wengle,1978,1979),smallintestine(Dewetal.,1980;Loofand 2000; Otterness and Weinshilboum, 1994). However, others Wengle,1979),andadrenalgland(AdamsandMcDonald,1981; demonstrated high expression of SULT2A1 mRNA in these Loof,1981).BA-sulfotransferaseactivitywasalsocharacterized organs (Dooley et al., 2000; Javitt et al., 2001). Other organs inotherspeciessuchashamsters(Barnesetal.,1979a),monkeys suchas,thebrainandovary,alsoexpressSULT2A1mRNAat (Barnesetal.,1986),mice(Takahashietal.,1990),rats(Chen moderate levels (Dooley et al., 2000; Javitt et al., 2001; etal.,1978a;Summerfieldetal.,1976;Takahashietal.,1990), Shimada et al., 2001). SULT2A1 Protein and enzyme-activity D andguineapigs(Chenetal.,1978b). data also confirmed the presence of SULT2A1 in the human ow n Several attempts were made to isolate and purify the BA- liver, stomach, and small intestine but not in the colon (Chen lo a SULT fractions from human livers (Chen and Segel, 1985; etal.,2003).Inhumanfetuses,SULT2A1proteinwasdetected de d Falanyetal.,1989;LoofandHjerten,1980;Radominskaetal., by immunohistochemistry in the adrenal cortex, liver, testis, fro 1990) and other species such as rats (Barnes and Spenney, and intestine, weakly detected inkidney, and was notdetected m 1982;Barnesetal.,1989;Chen,1981;Chenetal.,1977;Kane in lung, brain, heart, stomach, skeletal muscle, pancreas, or http Setpeanl.n,e1y9,881;98O2g)u.raSueltfaatli.o,n19o9f4),haynddroxhyamstesrteorisds(Bwaranseswaneldl slaprlgeeenam(Poaurknetsr oetfaDl.H,E19A9-4su).lfTatheetofetsaulpapdorretnpallagcelanntdalpersotrdougceens s://ac a characterized before the discovery of BA sulfation. Early on, biosynthesis. This is reflected by the high levels of SULT2A1 de m it was thought that BA and hydroxysteroid sulfation were expression in the fetal adrenal gland (Coughtrie, 2002). ic performed by distinct SULT isoforms. Evidence was then Based on the plethora of data available on the tissue .ou p providedthat,inhumans,theBA-SULTisthesameenzymeas distributionofSULT2A1mRNA,protein,andenzymeactivity, .c o the hydroxysteroid-SULT, which had been known for its high it can be concluded that the liver is the major site of BA m/to affinity for dehydroepiandrosterone (DHEA) (Radominska sulfation.However,these samedata alsosuggestthatBAs can x s et al., 1990). BA/DHEA-SULT was then cloned from human be sulfated in other organs such as the intestine. ci/a liver (Comer et al., 1993; Kong et al., 1992; Otterness et al., rtic 1992) and is currently known as SULT2A1. The crystal le-a s(Atrullcatlui-rHeasosfanhiuemt aanl., S2U00L7T;2LAin1dshayasetaalslo., 2b0e0e8n).dInetecromnitnraesdt SULFATION ASA BADETOXIFICATIONPATHWAY bstra IN VARIOUS SPECIES c tothepresenceofasingleSULT2Aenzymeinhumans,atleast t/1 0 8 2 isoforms of Sult2a have been cloned from mouse; mSult2a1 BAs are well known for their role in fat absorption. BAs /2 (Kong et al., 1993) and mSult2a2 (Kong and Fei, 1994), and form mixed micelles with phospholipids creating an emulsion /22 5 three isoforms have been cloned from rats; rSult2a1 (Ogura that aids in the intestinal absorption of fat and fat-soluble /1 6 et al., 1989), rSult2a2 (Ogura et al., 1990), and rSult2a3 vitamins (Hofmann and Hagey, 2008). Recently, BAs have 64 3 (Watabe et al., 1994). The various isoforms of Sult2a in rats been recognized as signaling molecules and hormones. BAs 8 3 appeartohavesimilarsubstratespecificity(Oguraetal.,1994). have been shown to regulate the expression of several target by The SULT2 family (hydroxysteroid SULTS) includes another genes, which play important roles in apoptosis, proliferation gu e subfamily of enzymes known as SULT2B, which does not (Jones et al., 1997; Sodeman et al., 2000), liver regeneration st o seem to have activity toward BA sulfation (Sakakibara et al., (Monte et al., 2002), as well as cholesterol, triglyceride n 2 1998; Shimada et al., 2002). (Kalaany and Mangelsdorf, 2006), energy (Watanabe et al., 7 M SULT2A1 has broad substrate specificity for endogenous 2006), glucose (Claudel et al., 2005), and their own a rc hydroxysteroid androgens, estrogens, and glucocorticoids homeostasis (Staudinger and Lichti, 2008). In addition, BAs h 2 (Chatterjee et al., 1994; Falany et al., 1995; Ogura et al., can be usedasbiomarkers for hepatobiliary diseases in infants 01 9 1994). SULT2A1 substrates also include BAs (Ogura et al., (Mills et al., 1998; Mushtaq et al., 1999), and to monitor the 1994), thyroid hormones (Li and Anderson, 1999), and xeno- functionsoforgansinvolvedintheirsynthesisandelimination, biotics such as tamoxifen (Shibutani et al., 1998), budesonide such as liver and kidney (Alme et al., 1977). BAs have also (Melocheetal.,2002),andbisphenols(Nishiyamaetal.,2002; been shown to play a role in the prevention of obesity and Paietal.,2002).Amongallsubstrates,DHEAandepiandroster- resistance to insulin, which can be a novel target to improve one have the highest affinity for Sult2a1 (Ogura et al., 1994). anddiagnosemetabolicdisorders(Libertetal.,1991;Tsukada DespiteitsroleinBAandandrogeninactivation,SULT2A1was et al., 1994). Furthermore, UDCA is used therapeutically in demonstrated to catalyze the formation of DNA- and protein- patients with gallstone and primary biliary cirrhosis (Poupon reactive metabolites of polycyclic aromatic hydrocarbons and et al., 1997). The physiologicroles of BAs have beenrecently benzylicalcohols(Glattetal.,1995;Falanyetal.,1995). reviewed (Hofmann and Hagey, 2008). BILEACIDSULFATION 231 In contrast to their physiologic functions, BAs are also Therefore,onlybasedonthephysicochemicalpropertiesofBA- cytotoxic.ThepathologiceffectsofBAsarebelievedtobedue sulfates,sulfationisexpectedtodecreaseBAtoxicity. totheirdetergent properties, whichenable them tobind toand Indeed,SulfationabolishesthecholestaticactivityofCDCA, solubilizemembranelipids(PlaaandPriestly,1976).BAscause CA,andDCA.Infact,thesulfatemetabolitesproduceacholeretic plasma and mitochondrial membrane disruption (Garner et al., effect(increasebileflow)inrats(EngandJavitt,1983;Yousef 1991;LeeandWhitehouse,1965;WeissmannandKeiser,1965; et al., 1987b). At the cellular level, sulfation decreases or Sanchez Pozzi et al., 2003). BAs exert a cathartic effect by abolishesLCAcytotoxicityagainsthepatocytes(Takikawaetal., altering water and salt transport in the colon (Mekhjian and 1991b)andvascularendothelialcells(Garneretal.,1991).Also Phillips, 1970). LCA and conjugates are potent pyrogens in sulfation reduces phospholipid biliary excretion, and prevents humans (Dillard and Bodel, 1970). BAs are also genotoxic, solubilization of the cellular membrane phospholipids (Yousef tumor promoters (Fukase et al., 2008; Rosignoli et al., 2008), et al., 1987b, 1992). Furthermore, sulfation decreases the car- D o andarebelievedtobeinvolvedincoloncancer(Narisawaetal., cinogenicity of LCA (Takahashi et al., 1993) and prevents the w n 1974; Reddy et al., 1976, 1977). The common factor between catharticeffectofCDCAandDCAonthecolon(Breueretal., lo a the various toxicities caused by BAs is that secondary BAs, 1983). In contrast to sulfation, G- or T-amidation does not de d LCA,andtoalesserextentDCAarethemosttoxic,whereasthe decrease the cholestatic and cytotoxic effects of BAs (Dawson fro primaryBAs,CA,andCDCA,arenotastoxic. andIsselbacher,1960;JavittandEmerman,1968). m h The pathologic effects on the liver were early recognized as Sulfation of LCA and T-LCA markedly decreases, but does ttp s a major toxicity of BAs (Holsti, 1956). It was then notpreventtheircholestaticactivity(Fisheretal.,1971;Yousef ://a demonstrated that LCA is the most hepatotoxic component of etal.,1981,1987b).Incontrast,G-LCA-sulfateisascholestatic c a d bile (Palmer, 1972). A variety of pathologic-hepatobiliary asG-LCAinrats(Fisheretal.,1971;Yousefetal.,1981).Itwas e m changes induced by LCA, including bile duct infarction, liver suggestedthatthedetoxificationeffectofsulfationonBAsmay ic .o fibrosis, and cirrhosis, were demonstrated in several species varyindifferentspeciesdependingontheprevalenceofT-orG- u p (Fickertetal.,2006;Hofmann,1999b;Palmer,1972).Amajor amidation(Yousefetal.,1981).Therefore,LCA-sulfateinduces .c o m manifestation of the hepatobiliary toxicity of BAs is cholesta- cholestasisinguineapigs,whichprimarilyconjugateBAwithG /to sis. Cholestasis is the decrease in bile flow and biliary BA (Yousefetal.,1981).FeedingguineapigswithaT-enricheddiet x s c excretion,whichultimatelycanleadtoacompletecessationof preventsthecholestaticeffectofG-LCA-sulfatebyinducingits i/a bile flow (Plaa and Priestly, 1976). All BAs have cholestatic conversiontoT-LCA-sulfate(Dorviletal.,1983).T-amidation rtic le effects at high doses. However, the cholestatic activity of BAs alone does not prevent the cholestatic effect of LCA, but -a b is proportional to their detergent properties, and is inversely T-amidationandsulfationcombineddoes(JavittandEmerman, s proportionaltothenumberofhydroxylgroupsonthesteroidal 1968).SulfationofLCAandT-LCA,butnotG-LCA,wasalso trac backbone, with LCA (mono-hydroxy BA) being the most shown to prevent the pathologic and morphologic effects that t/1 0 8 potent cholestatic agent (Baumgartner et al., 1992; Drew and LCAinflictsontheratliver(Leuschneretal.,1977;Yousefetal., /2 Priestly, 1979; Yousef et al., 1987b). LCA was demonstrated 1981). The difference in the toxicity of G-LCA- and T-LCA- /22 5 to induce cholestasis in several species including rats (Fisher sulfatesmaybeduetothefactthatalthoughsulfationincreases /1 6 et al., 1971; Javitt, 1966; Kakis and Yousef, 1978), hamsters LCA and T-LCA solubility, G-LCA-sulfate is not any more 64 3 (Schaffner and Javitt, 1966), and mice (Fickert et al., 2006). solublethanG-LCA(Careyetal.,1979).ThefactthatG-LCA- 8 3 ThehighcholestaticactivityofLCAisinpartattributedtoits sulfate is the major end-product metabolite of LCA in humans by g water insolubility. BAswith lowsolubilitymayform insoluble does not mean that sulfation cannot protect against G-LCA u e BAsalts,suchasCaþ2salts,whichprecipitateinthecanaliculi toxicity in humans. Even if G-LCA-sulfate is as cholestatic/ st o (Fickertetal.,2006;Yousefetal.,1997).Anothermechanismof hepatotoxicasG-LCA,sulfationisstillaneffectivepathwayfor n 2 liver toxicity is that BAs increase the biliary secretion of BAdetoxificationviaenhancingtheirfecalandrenalexcretion. 7 M phospholipidsuntilthehepatic phospholipidpool isexhausted. The most toxic BA, LCA, is a minor component (< 2%) of a rc Then, due to their detergent activity, BAs start to solubilize human bile and under normal conditions, does not exist in the h 2 membrane phospholipids, which disturbs the integrity of the enterohepatic system at levels high enough to produce any 01 9 canalicular membranes (Kakis and Yousef, 1978; Miyai et al., toxicity. However, LCA toxicity becomes relevant under 1977;SmallandAdmirand,1969;Yousefetal.,1987a).Several conditions of high exposure, such as in cholestasis and during molecularmechanismsunderlyingthecholestaticeffectofLCA CDCA or UDCA therapy. CDCA and UDCA decrease the have also been suggested (Beuers et al., 2003). Sulfate- cholesterol saturation of bile, and therefore, are used clinically conjugationaddsapermanentnegativechargetoBAsincreasing forgallstonedissolution(Danzingeretal.,1973).LCA,formed their water solubility and therefore, enhance their elimination by intestinal dehydroxlation, is the major metabolite of these (Oelberg et al., 1984). In addition, sulfation increases the BAs. Therefore, CDCA produces severe hepatic toxicity, as BA-micellar critical concentration, prevents cholesterol and a result of the accumulation of the LCA metabolite in rhesus phospholipid solubilization, and therefore, abolishes the BA- monkeys (Dyrszka et al., 1976; Gadacz et al., 1976), baboons detergent activity against membranes (Donovan et al., 1993). (Morrissey et al., 1975), and rabbits (Fischer et al., 1974). 232 ALNOUTI TABLE 1 ThePercentage ofIndividual andTotalBAs Presentinthe SulfateForm LCA DCA CDCA CA TotalBAs n References Healthysubjects Serum — 10.2 3.8 0 9 8 (Makinoetal.,1975) 85.7 0 7.7 0 13.2 10 (Campbelletal.,1975) 100 49.6 34.8 7.4 19.4 1 (Almeetal.,1977) 100 9.3 1.39 1.7 4.7 8 (Holzbachetal.,1980) 67 12 6.3 1.5 11.2 9 (Takikawaetal.,1982) 94 22 15 4 25 15 (Murataetal.,1983) D o 98.2 20.5 19.8 25.8 31.6 5 (SetchellandMatsui,1983) w n 63.9 12.8 5.8 6.6 10.7 36 (Takikawaetal.,1983) lo a 64 13 6 7 12 14 (Takikawaetal.,1986) d e Subje—ctswithcholestatic—diseases — — 16.8 56 (Katoetal.,1996) d fro m — 29.3 9.5 0 9.2 11 (Makinoetal.,1972) h 49.7 9 6.5 4.8 6.4 30 (Stiehl,1974a) ttp s — 39.3 18.5 0.2 12.1 8 (Makinoetal.,1975) ://a 67.5 0 9.3 18.3 22.9 10 (Campbelletal.,1975) c a — — — — <7.0 32 (vanBergeHenegouwenetal.,1976) d e 21–100 ?–100 ?–100 0–42 9.3–50 8 (Summerfieldetal.,1977) m — — — — <5.0 14 (Williamsetal.,1979) ic.o 100 55 25.5 2.5 7 (BremmelgaardandAlme,1980) up 100 63.6 28.9 17.6 26.6 3 (Capocacciaetal.,1981) .co 100 34.8 1.4 23.9 19 (Bartholomewetal.,1982) m 92 70 26 2 16 8 (Murataetal.,1983) /tox s 62.2 34.6 11.8 0.9 11 14 (Takikawaetal.,1983) c 79 86.7 28.5 4 30.2 4 (Takikawaetal.,1985) i/a 79.1 41.9 29.1 1.97 22.7 10 (Takikawaetal.,1986) rtic le — 15.1 17.4 12.6 — 29 (Stiehletal.,1990) -a — — — — 4–10.5 114 (Katoetal.,1996) bs Subjectswithcholestaticdiseases tra c Bile t/1 90 20 — — — 17 (Stiehletal.,1975) 08 — — — — <0.2 4 (Makinoetal.,1975) /2 /2 17–100 ?–100 1.3–5.1 0–18 2.1–4.4 8 (Summerfieldetal.,1977) 2 5 38.89 — — — — 10 (Stiehletal.,1978b) /1 6 0 0 0 0 0 14 (Williamsetal.,1979) 6 4 38.5 62 0.775 0.375 1.2 4 (Takikawaetal.,1985) 3 8 46.1 6.27 1.49 0.5 1.53 8 (Stiehletal.,1985) 3 b 52 — — — — 17 (Fisheretal.,1991) y g Healthysubjects u e Urine s — 56.27 100 — 64.5 3 (Makinoetal.,1975) t on 100 80 100 15 69.3 5 (Almeetal.,1977) 2 7 50 — — — — 10 (Stiehletal.,1978b) M a 97.89 69.66 85.9 1.18 59.8 7 (Takikawaetal.,1984) rc — — — — 37 3 (Mengetal.,1997) h 2 Subjectswithcholestaticdiseases 0 1 Urine 9 — — — — 57–93 2 (Makinoetal.,1972) — 97.1 92.9 31.5 73.9 8 (Makinoetal.,1974) 87.5 62.4 62.6 46.9 56.3 30 (Stiehl,1974a) 100 85 76 24 56 17 (Stiehletal.,1975) — 87.2 89.7 20.7 65.6 22 (Makinoetal.,1975) 81 54 69.8 27 52.2 20 (FrohlingandStiehl,1976) — — 80 26 60 32 (vanBergeHenegouwenetal.,1976) 68–100 36–100 36–100 0–64 59–79 8 (Summerfieldetal.,1977) 80.8 97 97.2 58.6 80.8 4 (Almeetal.,1977) — — — — 60 2 (Williamsetal.,1979) BILEACIDSULFATION 233 TABLE1—Continued LCA DCA CDCA CA TotalBAs n References — — 52 38 46.5 6 (Raedschetal.,1981) 100 56.4 35.8 0 26.5 10 (Capocacciaetal.,1981) 96.1 88.6 91.5 19.9 73.6 21 (Takikawaetal.,1984) 100 100 85.9 40.3 63.9 1 (HedenborgandNorman,1984) 99.5 68.5 87.3 23.5 74 4 (Takikawaetal.,1985) 87.5 73.3 56.6 22.7 35.4 29 (Stiehletal.,1990) 94.8 81.9 93.7 38.2 75.1 8 (Stiehletal.,1985) 89.3 69.2 76.5 35.9 94 (Shodaetal.,1990) — — — — 60 6 (Mengetal.,1997) D o w Note.ThepercentageofindividualBAs(LCA,DCA,CDCA,CA)presentinthesulfateformiscalculatedfromtheratioofBA-sulfate:BAconcentrationinall n lo itsforms.ThepercentageoftotalBAspresentinthesulfateformiscalculatedfromtheratiooftotalBA-sulfates:totalBAsinallitsforms.(n)Thenumberof a d humansubjectedtested. e d fro m h In humans, however, CDCA therapy is relatively safe, and is way in BDL rats, urinary excretion of CDCA-, T-CDCA-, ttp s not associated with hepatic injury (Schoenfield and Lachin, LCA-, and T-LCA- sulfates was less than 5% of the dose ://a 1981).ThedifferenceinCDCAtoxicityamongvariousspecies administered (Cleland et al., 1984; Little et al., 1991; Takada c a d is due to the ability of humans to sulfate the resulting LCA et al., 2003). Instead, most of the dose was retained in plasma e m metabolite, whichenhances LCAfecalexcretion,andprevents and peripheral tissues such as skin and muscle (Little et al., ic .o its accumulation in the enterohepatic system. Whereas, the 1991). The low urinary excretion of BA-sulfates in rats might u p other species lack such efficient-sulfating capabilities (Dew beduetothefactthatsulfationpreventshydroxylationofBAs. .c o m et al., 1982; Gadacz et al., 1976; Hofmann, 2004; Stellaard UnsulfatedBAsaremoreefficientlyhydroxylatedandexcreted /to et al., 1985). in urine than sulfated BAs. x s c Sulfation is a minor pathway of BA metabolism in rats and i/a mice. In rat primary hepatocytes, very low (Kirkpatrick and rtic le Belsaas,1985)orno(LambiotteandThierry,1980)BA-sulfating BA-SULFATES INHUMANSIN HEALTH ANDDISEASE -a b activitywasdetected.Furthermore,sulfationofLCAbyratliver s wasmorethana100-foldlessthanthatofhumans(Kirkpatrick DataonthelevelofBA-sulfatesvarymarkedly,inlargepart, trac et al., 1988). In addition, only small amounts to none of LCA becauseofthevarietyofanalyticaltechniquesusedtoquantify t/1 0 8 (Palmer,1971),CDCA,orCA(Clelandetal.,1984;Takitaetal., them.Table1summarizesthereportedproportionsoftotaland /2 1988) administered to rats were sulfated. Also, levels of individual BAs present in the sulfate form in human serum, /22 5 endogenous BA-sulfates in rat urine were undetectable or bile, and urine. The amount of total BAs excreted in urine is /1 6 constituted less than 0.001% of total BAs. In bile duct-ligated very low (< 1lM/day) under normal conditions. In various 64 3 (BDL) rats and mice, the undetectable orlow levelsof urinary occasions, it was reported that 40–70% of BAs in urine are 8 3 BA-sulfatesremainedunchanged(Leeetal.,2001)orincreased sulfated, Table 1. The concentration of total BAs in serum of by g to 0.2–2.7% of the total BAs in urine (Kinugasa et al., 1981; a healthy man is also very low (< 3lM), with less than 15% u e Marschall et al., 2006; Takita et al., 1988). Instead, hydroxyl- present in the sulfated form, Table 1. Wide inter-individual st o ation at the 6b position represents the major pathway of BA variations also exist in the levels of BA-sulfates in serum, n 2 detoxificationinrats,whichisinducedincholestaticconditions where proportions as high as 64% were reported in some 7 M to enhance the urinary excretion of accumulated BAs (Dan- healthy individuals. Very low proportions of BAs in bile are a rc ielsson,1973;Greimetal.,1972b;Kinugasaetal.,1981;Takita sulfated, with available data ranging from undetectable up to h 2 etal.,1988).Similartosulfationinhumans,hydroxylationalso 4%. From Table 1, it can also be seen that most BAs in urine, 01 9 protects against BA toxicity in rodents (Bagheri et al., 1978; small proportions ofserum BAs, and negligibleproportionsof Hunt et al., 1964; Takikawa et al., 1997a). Other reports biliary BAs are sulfated. Furthermore, a common finding of however,demonstratedthecapabilityofrodentstosulfateBAsto these studies is that the proportion of individual BAs that are a significant extent under cholestatic conditions (Leuschner sulfateddecreaseswiththeincreaseinthenumberofhydroxyl etal.,1977).Furthermore,laterreportsdemonstratedthat5%of groupson theBA.Therefore,theLCA (mono-hydroxy BA)is serumand40%ofurinaryBAsarepresentinthesulfateformin almost exclusively present in the sulfated form, whereas CA rats(Puruckeretal.,2001). (tri-hydroxy BA) is mostly present in the unsulfated form. Rats do not only have lower BA sulfation capabilities Structuralfactors,inadditiontothenumberofhydroxylgroups, compared with humans, but they are also less capable of also play a role in determining the extent of sulfation. The eliminating BA-sulfates. Despite the obstructed biliary path- percentage of DCA present in the sulfated form is generally 234 ALNOUTI higher than that of CDCA, despite the fact that they are both Therefore, it can be concluded that the increase in the dihydroxy BAs. Despite the high proportion of LCA present in formationandurinaryexcretionofBA-sulfatesduringcholestatic thesulfateform,theproportionoftotalBA-sulfatesremainsvery diseasesisunlikelytobeduetoenhancedSULT2A1activityor lowinserumandbile,becauseLCA,inallofitsforms,doesnot expression,butrather,duetotheincreaseinthesubstrate(BAs) constitute more than 2% of total BAs (Perwaiz et al., 2001). availabilityforsulfation(Barnesetal.,1979b;Galleetal.,1989; In hepatobiliary/cholestatic diseases, due to the impairment KirkpatrickandBelsaas,1985).Itisalsopossiblethatsulfationof of biliary excretion, urinary excretion of BAs increases more BAs is enhanced as an adaptive/compensatory mechanism at than100fold.Similartothesituationundernormalconditions, early stages of cholestasis but, with additional damage at alargeproportionofBAs(25–80%)inurineisexcretedinthe advancedstages,thelivercapabilitytosulfateBAsisdecreased sulfated form under cholestatic conditions, Table 1. The (GaleazziandJavitt,1977;Stiehletal.,1978a). amount of BA-sulfates excreted in urine was suggested to be D o used as a specific biomarker for the diagnosis of intrahepatic GENDERDIFFERENCES IN BASULFATION w n cholestasisinpregnantwomen(Huangetal.,2007).Underthe lo a same pathologic conditions, serum BA levels also increase GenderdifferenceswereobservedinrelationtoBAsulfation de d however, the concentration of sulfated BAs does not increase in several animal species. In germfree rats, the percentage of fro in parallel with the increase in unsulfated BAs in the serum. BA-sulfates was 10-fold higher in female cecal content and m h This indicates the efficient renal excretion of BA-sulfates, feces compared with males (Eyssen et al., 1977; Parmentier ttp s whereBA-sulfatesaremoreefficientlyclearedfromseruminto et al., 1981). In female rats, the percentage of BA-sulfates in ://a to urine than unsulfated BAs. Therefore, the percentage of bile is about 1%, whereas less than 0.2% of biliary BAs are c a d sulfated BAs in serum either remains unchanged, or even sulfated in males (Eriksson et al., 1978). e m decrease, with hepatobiliary diseases in humans. Other studies At the enzyme-activity level, livers from female rats have ic .o however,haveindicatedthattheproportionofsulfatedBAsin threefoldhigherSult-activitytowardT-LCAthanthatinmales u p serum in hepatobiliary diseases increases up to 40% of total (Hammerman et al., 1978). In hamsters, female livers have .c o m BAs. In cholestatatic patients, sulfated BAs in bile remains a four-fold higher Sult activity toward G-CDCA than that in /to low,andranges from undetectableupto4%oftotalBAs. The males,thishigherenzymaticactivityhoweverisnotreflectedon x s c Percentage ofBA-sulfates inthe human liverwas estimated to the percentage of BA-sulfates in bile, urine, or blood (Barnes i/a be less than 10%, which further decreases under cholestatic etal.,1979a,b).Inmice,a70-foldhigher Sultactivity toward rtic le conditions (Fischer et al., 1996; Greim et al., 1972a). DHEAwasdemonstratedinliversfromfemalescomparedwith -a b Due to the impairment of the biliary excretion route under that in males (Borthwick et al., 1995a). The gender-specific s cholestaticconditions,theamountofBAsincludingBA-sulfates Sult2a1 activity and expression were shown to be due to trac increasesinserum,tissues,andurine.However,theratioofBA- stimulatoryeffectsbyfemaleestrogens,andsuppressiveeffects t/1 0 8 sulfates:total BAs remains constant or may decrease. This may by male androgens and male-pattern growth hormone (Borth- /2 be ascribed to the impairment of the liver capability to sulfate wick et al., 1995b; Carlstedt-Duke and Gustafsson, 1973; /22 5 BAs as a result of the deteriorated liver function under these Collinsetal.,1987;Hammermanetal.,1978;Kaneetal.,1984; /1 6 pathologic conditions (Fischer et al., 1996; Kato et al., 1996; Kirkpatricketal.,1985;Labrieetal.,1994;LiuandKlaassen, 64 3 Murataetal.,1983;Takikawaetal.,1983).Itwasthoughtthat 1996; Torday et al., 1971; Yamazoe et al., 1987). The sex 8 3 the increase in the amount of BA-sulfates excreted in urine differences start at puberty, where Sult2a1 activity begins to by g might be due to a compensatory increase in BA sulfation as decline while female’s stays constant or does not decrease as u e a result of the upregulation of the hepatic SULT2A activity/ fast(Balistrerietal.,1984;Chenetal.,1982;Kaneetal.,1988). st o expression during cholestatic conditions. However, it was then However,thegenderdifferencesinSult-activitytowardT-LCA n 2 demonstratedthathepaticSULTactivitytowardBAsandother was liver specific, and there were no gender differences in 7 M substrates(Iqbaletal.,1990;LoofandNyberg,1983;Loofand kidney activity (Hammerman etal., 1978). a rc Wengle, 1982), and SULT2A1 protein expression (Elekima This gender difference also exists at the Sult mRNA and h 2 et al., 2000) are actually reduced during cholestatic diseases in proteinlevels.Inrats,Sult2a1mRNAlevels are atleast6-fold 01 9 humans. Others, however, reported that SULT2A1 mRNA higherinfemalethanmalelivers(Chatterjeeetal.,1987;Dunn expression was not altered in humans in cholestatic diseases and Klaassen, 1998; Runge-Morris and Wilusz, 1991). Also, (Zollner et al., 2007). In animal models, hepatic BA-SULT a 15-fold higher Sult2a1 protein expression was reported in activity and the percentage of BA-sulfates were shown to be livers from female rats than that in males (Chen et al., 1995; reduced in BDL-hamsters (Barnes et al., 1979b) and in BDL- Hommaetal.,1992).Inmice,themRNAexpressioninfemale pregnant rats (Chen et al., 1982). Furthermore, cholestasis liversismuchhigherthanthatinmales(AlnoutiandKlaassen, inducedbyLCAadministrationdidnotinduceBA-Sultactivity 2006, 2008; Saini et al., 2004; Wu et al., 2001). in rats (Balistreri et al., 1984). Also both LCA and BDL Sult2a1 expression is also age-dependent. Sult2a1 mRNA suppressed Sult2a1 expression in female mice (Uppal et al., levels are very low in livers obtained from infant rats before 2007). and right after birth. The expression starts increasing until