Appearance of atypical 3α,6β,7β,12α-tetrahydroxy-5β-cholan-24-oic acid in spgp knockout mice

Bile formation and its canalicular secretion are essential functions of the mammalian liver. The sister-of-p-glycoprotein (spgp) gene was shown to encode the canalicular bile salt export protein, and mutations in spgp gene were identified as the cause of progressive familial intrahepatic cholestasis type 2. However, target inactivation of spgp gene in mice results in nonprogressive but persistent cholestasis and causes the secretion of unexpectedly large amounts of unknown tetrahydroxylated bile acid in the bile. The present study confirms the identity of this tetrahydroxylated bile acid as 3α,6β,7β,12α-tetrahydroxy-5β-cholan-24-oic acid. The data further show that in serum, liver, and urine of the spgp knockout mice, there is a significant increase in the concentration of total bile salts containing a large amount of tetrahydroxy-5β-cholan-24-oic acid. The increase in total bile acids was associated with up-regulation of the mRNA of cholesterol 7α-hydroxylase in male mice only.It is suggested that the lower severity of the cholestasis in the spgp knockout mice may be due to the synthesis of 3α,6β,7β,12α-tetrahydroxy-5β-cholan-24-oic acid, which neutralizes in part the toxic effect of bile acids accumulated in the liver. Bile formation and its canalicular secretion are essential functions of the mammalian liver. The sister-of-p-glycoprotein (spgp) gene was shown to encode the canalicular bile salt export protein, and mutations in spgp gene were identified as the cause of progressive familial intrahepatic cholestasis type 2. However, target inactivation of spgp gene in mice results in nonprogressive but persistent cholestasis and causes the secretion of unexpectedly large amounts of unknown tetrahydroxylated bile acid in the bile. The present study confirms the identity of this tetrahydroxylated bile acid as 3α,6β,7β,12α-tetrahydroxy-5β-cholan-24-oic acid. The data further show that in serum, liver, and urine of the spgp knockout mice, there is a significant increase in the concentration of total bile salts containing a large amount of tetrahydroxy-5β-cholan-24-oic acid. The increase in total bile acids was associated with up-regulation of the mRNA of cholesterol 7α-hydroxylase in male mice only. It is suggested that the lower severity of the cholestasis in the spgp knockout mice may be due to the synthesis of 3α,6β,7β,12α-tetrahydroxy-5β-cholan-24-oic acid, which neutralizes in part the toxic effect of bile acids accumulated in the liver. Bile acids are synthesized from cholesterol in the liver and secreted into the bile duct via an active process in the canaliculus. This process is mediated by an ATP-dependent system, sister of p-glycoprotein (spgp), which is also known as bile salt-exporting pump (BSEP) (1Vlahcevic Z.R. Pandal W.M. Todd Stravitz R. Regulation of bile acid biosynthesis.Gastroenterol. Clin. North Am. 1999; 28: 1-25Google Scholar, 2Muller M. Jansen P.L.M. Molecular aspects of hepatobiliary transport.Am. J. Physiol. 1997; 272: G1285-G1303Google Scholar, 3Gerloff T. Stieger B. Hagenbuch B. Madon J. Landmann L. Roth J. Hofmann A.F. The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver.J. Biol. Chem. 1998; 273: 10046-10050Google Scholar, 4Muller M. Ishikawa T. Berger U. Klunemann C. Lucka L. Schreyer A. Kannicht C. Reutter W. Kurz G. Keppler D. ATP-dependent transport of taurocholate across the hepatocyte canalicular membrane mediated by a 110-kDa glycoprotein binding ATP and bile salt.J. Biol. Chem. 1991; 266: 18920-18926Google Scholar, 5Nishida T. Gatmaitan Z. Che M. Arias I.M. Rat liver canalicular membrane vesicles contain an ATP-dependent bile acid transport system.Proc. Natl. Acad. Sci. USA. 1991; 88: 6590-6594Google Scholar). A defect in the human SPGP gene has been suggested to be the basis for type 2 progressive familial intrahepatic cholestasis (PFIC2) (6Strautnieks S.S. Bull L.N. Knisely A.S. Kocoshis S. Dahl N. Arnell H. Sokal E. Dahan K. Childs S. Ling V. Tanner M.S. Kagalwalla A.F. Nemeth A. Pawlowska J. Baker A. Mielivergani G. Freimer N.B. Gardiner R.M. Thompson R.J. A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis.Nat. Genet. 1998; 20: 233-238Google Scholar). Recently, the mouse spgp was cloned and Spgp knockout mice were generated (7Wang R. Salem M. Yousef I.M. Tuchweber B. Lam P. Childs S.J. Helgason C.D. Ackerley C. Phillips M.J. Ling V. Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis.Proc. Natl. Acad. Sci. USA. 2001; 1398: 2011-2016Google Scholar). In these mice, the target disruption of the Spgp gene showed reduction in biliary excretion of bile salts and impaired bile flow; however, mutant mice did not display the expected progressive cholestatic phenotype seen in human PFIC2 (7Wang R. Salem M. Yousef I.M. Tuchweber B. Lam P. Childs S.J. Helgason C.D. Ackerley C. Phillips M.J. Ling V. Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis.Proc. Natl. Acad. Sci. USA. 2001; 1398: 2011-2016Google Scholar). It is suggested that alteration in bile acid metabolism, especially the production of a tetrahydroxylated bile acid, may alleviate the deleterious effects of possible accumulation of high bile salts in the liver of Spgp mutated mice. Therefore, the objective of this study is to characterize the biliary, liver, plasma, and urinary bile acids in the Spgp mutated mice. The data confirm for the first time the structure of the tetrahydroxylated bile acid as 3α,6β,7β,12α-tetrahydroxy-5β-cholan-24-oic acid, and further show a significant increase in the concentration of total bile salts in liver, plasma, and urine of the mutant mice that contain a large amounts of this tetrahydroxylated bile acid. Despite the increase in the total bile acids in the liver of both male and female spgp knockout mice, the expression of the mRNA for cholesterol 7α-hydroxylase was significantly up-regulated in the male mice but not affected in the females. These data suggest that the expression of cholesterol 7α-hydroxylase could not explain the changes in the bile acids pool in the spgp knockout mice. Glycine- and taurine-conjugated cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, 5β-cholanic acid, and 3α,12α,diol 7-one 5β-cholanic acid were obtained from Calbiochem, San Diego, CA, and were of at least 98% purity. Muricholic acid was purchased form Steraloid Inc, Wilton, NH. Two pure reference compounds of tetrahydroxylated bile acids, namely 3α,6β,7β,12α-tetrahydroxy-5β-cholan-24-oic acid and 1β, 3α,7α,12α-tetrahydroxy-5β-cholan-24-oic acids, were gifts from Prof. Taiju Kuramoto, Institute of Pharmaceutical Sciences, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-Ku, Hiroshima, 734, Japan. Octadecyl (C18) Baker bond (R) extraction column was obtained from J. J. Baker, Inc. All other solvents and chemicals used were of either HPLC grade or of known analytical purity obtained from Sigma-Aldrich Chemical Co. Reagents for the Northern blotting of cholesterol 7α-hydroxylase mRNA were purchased from Invitrogen Canada, Inc, Burlington, ON; Amersham Canada, Ltd., Oakville, ON; and Qiagen Canada Inc., Mississauga ON. Gallbladder bile, serum, liver, and urine samples were obtained from homozygous, heterozygous, and the wild-type male and females of spgp knockout mice. The methodology used for the determination of total and individual bile acids in bile, serum, and urine was similar to that described previously by this laboratory (8Perwaiz S. Tuchweber B. Mignault D. Yousef I.M. Determination of bile acids in biological fluids by liquid chromatography-electrospray tandem-mass spectrometry.J. Lipid Res. 2001; 42: 114-119Google Scholar). One microgram of internal standard (3,12 diol 7-one 5β-cholanic acid) was added to each sample of serum (0.5 ml), urine (50–100 μl), and bile (20 μl), followed by the addition of 0.1 M sodium hydroxide (2 ml) to each sample, then heated at 64°C for 5–10 min. These samples were then subjected to a solid-phase extraction using a Bond-Elute C18 cartridge. The C18 cartridge was preconditioned prior to loading the samples with successive elution of 2 ml of chloroform-methanol (2:1, v/v), methanol, and HPLC water solutions. After loading the samples, the column was washed with 2 ml of HPLC water and n-hexane. The column was left for 10 min to remove any excess solvents. Bile acids were recovered from the cartridge by elution with methanol (5 ml). One part of the eluent was then evaporated to dryness under nitrogen. The residue was dissolved in 1 ml of acetonitrile-water (1:1, v/v) for the electrospray-tandem mass spectrometry (ES-MS/MS) analysis. The other portion was hydrolyzed and derivatized for gas chromatography mass-spectrometry (GC-MS) analysis. The bile acids from the liver tissues were extracted in a similar manner from the homogenate of 1 g of liver tissues in saline to which 10 μg of the internal standard was added. A Hewlett Packard LC system (HPLC series 1100) equipped with automatic sample injector and was connected to a Quattro electrospray tandem mass spectrometer (Micromass). The LC system was used only for injecting the samples and was operated isocratically at flow rate of 10 μl/min. Liquid nitrogen was used as nebulizer and argon as collision gas. Negative ion mass spectra of the elutes were recorded in the mass range of 300–800 amu. Multiple reaction-monitoring mode was used for the identification and quantification of bile acids. In this mode, both the MS1 and MS2 are used together, but in static mode, MS1 is set to transmit only the parent mass of interest, which is fragmented in the collision cell. Specific fragment ions obtained are selected and analyzed in the second spectrometer (MS2). To obtain consistent fragmentation for MS2, the collision offset voltage and collision energy were optimized to 25V and 50V, respectively. The molecular and daughter ions selected for the mass spectrometers MS1 and MS2 for each glycine-conjugated bile acid (tetra-, tri-, di-, and monohydroxylated bile acids) were: m/z 480.6 and 74; 464.6 and 74; 448.6 and 74; and 432.6 and 74 respectively; for taurine-conjugated bile acids (tetra-, tri-, di-, and monohydroxylated) were: m/z 530.8 and 124; 514.8 and 124; 498.8 and 124; and 482.6 and 124, respectively; and for the internal standard (3,12 diol 7-one 5β-cholanic acid) were m/z 405.6 and 123. Quantification was made using Micromass Mass lynx 3.0 software. Standard bile acids were processed and analyzed in a similar manner as described above. The methodology used for determination of total and individual bile acids was similar to that previously described (8Perwaiz S. Tuchweber B. Mignault D. Yousef I.M. Determination of bile acids in biological fluids by liquid chromatography-electrospray tandem-mass spectrometry.J. Lipid Res. 2001; 42: 114-119Google Scholar). In this method, 5β-cholanic acid (100 μg) was added as internal standard to each sample (gallbladder bile, serum, liver, and urine tissue) and then extracted by octadecyl (C18) cartridge as described before in the preparation of the samples for ES/MS/MS analysis. The extracted bile acids were then evaporated to dryness under nitrogen, and hydrolyzed in 2.5 N NaOH at 150–160°C overnight. Bile acids were then extracted methylated and acetylated. Identification and quantification of the bile acids were achieved by GC/MS using a Hewlett-Packard 5890 gas chromatography equipped with a Hewlett-Packard 5971A mass selective detector employing the selected ion-monitoring mode. In this method, the selected ions for the different bile acids were for 3α,6β,7β,12α-tetrahydroxy-5β-cholan-24-oic acid (m/z 444 and 384), cholic acid (m/z 253 and 368), chenodeoxycholic acid (m/z 255 and 370), deoxycholic acid (m/z 255 and 370), lithocholic acid, (m/z 257 and 372), and 5β-cholanic acid, (m/z 217 and 374). Quantification was carried out using a correction factor obtained by using 5β-cholanic acid as internal standard. Bile acid standards were processed and analyzed in a similar manner as samples. Total RNA was isolated from frozen mouse liver using the TRIzol extraction (Invitrogen). Using the Qiagen Oligotex midi mRNA extraction kit, mRNA was isolated from total RNA. The mRNA sample was diluted 1:4 with denaturing buffer (500 μl formamide, 180 μl formaldehyde, 100 μl 10× MOPs buffer (0.4 M MOPS, pH 7.0, 0.1 M sodium acetate, 0.01 M EDTA) to a total volume of 12 μl and heated for 15 min at 55–60°C. After being heated, 3 μl loading buffer (50% v/v) glycerol (1 mM EDTA, pH 8.0, 0.25% w/v), bromophenol blue, and 0.25% (w/v) xylene cyanol FF were added to each sample. After electrophoresis, RNA was transferred to a nylon membrane (Hybond-N+, Amersham). DNA probe template of cholesterol 7α-hydroxylase (748 bp, nt 675–1423 in NM_007824) was PCR amplified and purified using the Quiex II kit (Qiagen). The probe was labeled using Random Priming (Invitrogen), and labeled probe was purified using a SephaDex-G50 column. The blots were hybridized at 65°C. All values represent mean and standard deviation of six or more in each the were analyzed by an was In the heterozygous, and the type mice, the bile salts were with and was with In 1 also that in the wild-type and mice, more of the bile salts were bile salt in both the The showed that cholic acid for of the bile acids and the is and acids, with being the bile acid was in the bile salt obtained the male and female of the wild-type and In the the biliary bile acid as the bile salts was to in and in females and the acid the bile acid The spectra show ions molecular of to the molecular of the bile The spectra obtained by that this is tetrahydroxylated bile acid, as by the of an ion at m/z and of the tetrahydroxylated bile acids This bile acid for in and in females of bile acids in the bile of spgp knockout male female spgp male female spgp male female spgp male female spgp male female spgp male female spgp male female spgp male female spgp not shown are of and di-, tri-, and tetrahydroxylated bile acids. Bile acids were extracted from μl of bile. represents the of individual for six male female spgp in a spectra ion obtained from bile of wild-type and of spgp knockout mice. Bile acids were extracted from μl of bile. The the of bile acids in bile. The molecular ions at m/z and 530.8 to of di-, tri-, and tetrahydroxylated bile acids, respectively. The other ions present in the different spectra not to bile spectra of the of tetrahydroxylated bile acid present in the bile, serum, and liver of the spgp knockout mice obtained by spectra of the of standard of 3α,6β,7β,12α-tetrahydroxy-5β-cholan-24-oic acid obtained by not shown are of and di-, tri-, and tetrahydroxylated bile acids. Bile acids were extracted from μl of bile. represents the of individual for six to bile acids, the bile acid in the liver was with The bile acid concentration of liver tissue) was significantly to in the mice in both and type in both to bile, bile acid was the bile acid in wild-type mice and was significantly to in and in females of the mice. acid was the bile acid in the wild-type and mice, acid was the bile acid in the mice. bile acids for in and in females of the mice of bile acids in the liver homogenate of spgp knockout male female spgp male female spgp male female spgp male female spgp male female spgp male female spgp male female spgp male female spgp male female spgp male female spgp male female spgp male female spgp male female spgp male female spgp male female spgp male female spgp 2 the bile acid in the liver homogenate of spgp knockout mice. Bile acids were extracted from 1 g of liver shown are of di-, tri-, and tetrahydroxylated bile acids. represents of individual for six male female spgp in a 2 the bile acid in the liver homogenate of spgp knockout mice. Bile acids were extracted from 1 g of liver shown are of di-, tri-, and tetrahydroxylated bile acids. represents of individual for six The of of the serum bile acids was different from both the bile and the liver bile acids. In the wild-type and mice, the for from to of the total bile acids in both the The of glycine-conjugated bile acids was to in the mice. bile acids for and in and females of mice, The total bile acids were significantly in the mice as with type and in both the however, the increase was in females as with to bile acid there was an increase in the of bile acids in the type and as with the in the bile and the liver In the spgp mice, there was a reduction in the of and bile acids that could be for by an increase in the of tetrahydroxylated bile of bile acids in the serum of spgp knockout male female spgp male female spgp male female spgp male female spgp male female spgp male female spgp male female spgp male female spgp male female spgp 3 represents the bile acid in the serum of spgp knockout mice. Bile acids were extracted from ml of shown are of and di-, tri-, and tetrahydroxylated bile acids. represents of individual for six male female spgp in a 3 represents the bile acid in the serum of spgp knockout mice. Bile acids were extracted from ml of shown are of and di-, tri-, and tetrahydroxylated bile acids. represents of individual for six The of urinary bile acids was also different from the and liver bile acid however, was similar to serum bile acids. In the the bile acids for and in the male and female mice, The of glycine-conjugated bile acids in mice was similar in females but in However, in the the urinary bile acids were with and in the and The of the bile acids was and in and and respectively. However, in the the of of the bile acids was The bile acids in the were of the taurine-conjugated and tetrahydroxylated bile acids bile acids in spgp knockout male female spgp male female spgp male female spgp male female spgp male female spgp male female spgp male female spgp male female spgp shown are of and di-, tri-, and tetrahydroxylated bile acids. represents of individual for six male female spgp in a shown are of and di-, tri-, and tetrahydroxylated bile acids. represents of individual for six was a increase in the of mRNA of cholesterol in the male of mice in to however, in the a reduction in the expression of the mRNA of cholesterol was This study represents the first the bile acid in bile, liver serum, and urine of spgp knockout mice that the bile acid pump in the canalicular The results as a significant increase in liver, serum, and urinary bile acids in the mice, the that disruption of the canalicular to associated with cholestasis (7Wang R. Salem M. Yousef I.M. Tuchweber B. Lam P. Childs S.J. Helgason C.D. Ackerley C. Phillips M.J. Ling V. Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis.Proc. Natl. Acad. Sci. USA. 2001; 1398: 2011-2016Google Scholar). However, the is the biliary secretion of identified tetrahydroxylated bile acid acid) as as its in liver serum, and urine of the mice. was also a significant bile acids of the male and female mice, an the gene disruption and other of Scholar). The data obtained by the the molecular of the compounds present in the biological sample, and is not possible to the different di-, tri-, and bile acids. However, is possible to the different of these bile acids. The the other identification and quantification of the different di-, tri-, and bile acids. However, the preparation of the sample for the the of the acid to obtain the of of the different bile acids in the samples. both is a of the for the identification and quantification of the bile The biliary bile acids in mice are of taurine-conjugated cholic acid and acids, with glycine-conjugated bile acids or bile acids L. V. H. R. B. W. S. M. Jansen P. associated with bile in a mouse of 1999; Scholar). The data obtained in this study confirm this in the wild-type and mice, that any changes in bile acid in the type be due to the disruption of the spgp In the mice, there was an increase in the of acids and the of previously tetrahydroxylated bile acid The of the tetrahydroxylated bile acids has been before in intrahepatic cholestasis of bile acids in and in cholestasis of J. Clin. Scholar). However, the structure of the tetrahydroxylated bile acid before and obtained from reference compounds to from the structure obtained for the seen in this study tetrahydroxylated bile acids were also in cholestasis and R. L. T. J. T. Identification of acid in human of in bile acid in Lipid Res. Scholar, A. B. excretion of tetrahydroxylated bile acids in with and J. Clin. Scholar). However, of the ions of these compounds were present in The of the of the tetrahydroxylated bile acid obtained in this study is similar to the spectra obtained from the 3α,6β,7β,12α-tetrahydroxy-5β-cholan-24-oic acid standard The spectra an ion at m/z the of the This is further by the of different fragment ions at m/z 444 and by the of and from the molecular ion (m/z The of the the is further by the in which the molecular of these bile acids was or more the molecular of the bile acids. To further the identification of this tetrahydroxylated bile acid, the time of the of acid was to the time of bile acid. It is that the of the bile acids in part the of in the bile acids, and tetrahydroxylated bile acids could be more bile acids membrane disruption by bile Res. 1998; Scholar). This may be the that this mutant mouse is to in part for the of high liver and serum bile acids and could represent a the disruption of the spgp gene in mice and in In this disruption was associated with however, in these mice, this disruption to persistent but not progressive cholestasis (7Wang R. Salem M. Yousef I.M. Tuchweber B. Lam P. Childs S.J. Helgason C.D. Ackerley C. Phillips M.J. Ling V. Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis.Proc. Natl. Acad. Sci. USA. 2001; 1398: 2011-2016Google Scholar). The in the structure of the tetrahydroxylated bile acids in different human cholestasis and in the mice may the in the of the by different The structure of tetrahydroxylated acid) in this study is in with the bile acid synthesis in these mice. The high concentration of cholic acid in the type an active The of chenodeoxycholic acid and the of acid an active of chenodeoxycholic acid or its lithocholic acid in the liver of the is possible that the process by the of high bile acid and the synthesis of acid. It has been suggested that of the bile acids pool could in up-regulation of cholesterol 7α-hydroxylase and bile acid synthesis S.J. G. T. Regulation of cholesterol and bile acid in J. 1991; Scholar). data confirm this in mice, only further the spgp and females In of the bile acid in the bile, liver, serum, and urine confirm the identity of tetrahydroxylated bile acid previously to be in the spgp knockout The data further suggest that the of the liver of the mutant Spgp mice by high concentration of bile acids from the disruption of the may be in part by the production of the tetrahydroxylated bile acid. the biliary secretion of acid and tetrahydroxylated bile acids in the spgp knockout mice the of bile acid transporter or for the secretion of these bile acids in the mice T. of bile acid transport in a by of for bile acid and Biol. Chem. 1997; 272: Scholar, T. H. of the transport of cloned 3 Biol. Chem. 1999; Scholar, K. H. acid of results in transport for J. Physiol. Physiol. 2001; Scholar, S. R. P. Arias I.M. a to the canalicular of the J. Physiol. 1999; Scholar). This was by a from the of Canada and

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