FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2088794057> ?p ?o ?g. }

• W2088794057 endingPage "18666" @default.
• W2088794057 startingPage "18658" @default.
• W2088794057 abstract "Peroxisomal β-oxidation is an essential step in bile acid synthesis, since it is required for shortening of C27-bile acid intermediates to produce mature C24-bile acids. d-Bifunctional protein (DBP) is responsible for the second and third step of this β-oxidation process. However, both patients and mice with a DBP deficiency still produce C24-bile acids, although C27-intermediates accumulate. An alternative pathway for bile acid biosynthesis involving the peroxisomal l-bifunctional protein (LBP) has been proposed. We investigated the role of LBP and DBP in bile acid synthesis by analyzing bile acids in bile, liver, and plasma from LBP, DBP, and LBP:DBP double knock-out mice. Bile acid biosynthesis, estimated by the ratio of C27/C24-bile acids, was more severely affected in double knock-out mice as compared with DBP–/– mice but was normal in LBP–/– mice. Unexpectedly, trihydroxycholestanoyl-CoA oxidase was inactive in double knock-out mice due to a peroxisomal import defect, preventing us from drawing any firm conclusion about the potential role of LBP in an alternative bile acid biosynthesis pathway. Interestingly, the immature C27-bile acids in DBP and double knock-out mice remained unconjugated in juvenile mice, whereas they occurred as taurine conjugates after weaning, probably contributing to the minimal weight gain of the mice during the lactation period. This correlated with a marked induction of bile acyl-CoA:amino acid N-acyltransferase expression and enzyme activity between postnatal days 10 and 21, whereas the bile acyl-CoA synthetases increased gradually with age. The nuclear receptors hepatocyte nuclear factor-4α, farnesoid X receptor, and peroxisome proliferator receptor α did not appear to be involved in the up-regulation of the transferase. Peroxisomal β-oxidation is an essential step in bile acid synthesis, since it is required for shortening of C27-bile acid intermediates to produce mature C24-bile acids. d-Bifunctional protein (DBP) is responsible for the second and third step of this β-oxidation process. However, both patients and mice with a DBP deficiency still produce C24-bile acids, although C27-intermediates accumulate. An alternative pathway for bile acid biosynthesis involving the peroxisomal l-bifunctional protein (LBP) has been proposed. We investigated the role of LBP and DBP in bile acid synthesis by analyzing bile acids in bile, liver, and plasma from LBP, DBP, and LBP:DBP double knock-out mice. Bile acid biosynthesis, estimated by the ratio of C27/C24-bile acids, was more severely affected in double knock-out mice as compared with DBP–/– mice but was normal in LBP–/– mice. Unexpectedly, trihydroxycholestanoyl-CoA oxidase was inactive in double knock-out mice due to a peroxisomal import defect, preventing us from drawing any firm conclusion about the potential role of LBP in an alternative bile acid biosynthesis pathway. Interestingly, the immature C27-bile acids in DBP and double knock-out mice remained unconjugated in juvenile mice, whereas they occurred as taurine conjugates after weaning, probably contributing to the minimal weight gain of the mice during the lactation period. This correlated with a marked induction of bile acyl-CoA:amino acid N-acyltransferase expression and enzyme activity between postnatal days 10 and 21, whereas the bile acyl-CoA synthetases increased gradually with age. The nuclear receptors hepatocyte nuclear factor-4α, farnesoid X receptor, and peroxisome proliferator receptor α did not appear to be involved in the up-regulation of the transferase. Bile acids play an important role in the solubilization and digestion of dietary lipids and in the excretion of cholesterol. Bile acids are synthesized from cholesterol in the liver via a series of reactions involving many different enzymes located throughout the cell (1Russell D.W. Annu. Rev. Biochem. 2003; 72: 137-174Crossref PubMed Scopus (1406) Google Scholar). The nonpolar steroid nucleus of cholesterol is converted into a considerably more polar steroid by enzymes located in the endoplasmic reticulum and cytosol. In the mitochondrion, a carboxyl group is formed on the aliphatic side chain of the sterol molecule, which is shortened by β-oxidation in the peroxisome (Fig. 1). As a consequence, bile acid synthesis is disturbed in peroxisome biogenesis disorders and also in peroxisomal DBP 1The abbreviations used are: DBP, d-bifunctional protein; ACOX1, straight-chain acyl-CoA oxidase; ACOX2, trihydroxycholestanoyl-CoA oxidase; ACOX3, pristanoyl-CoA oxidase; AMACR, α-methylacyl-CoA racemase; BACS, bile acyl-CoA synthetase; BAAT, bile acyl-CoA:amino acid N-acyltransferase; CA, cholic acid; CDCA, chenodeoxycholic acid; DHCA, dihydroxycholestanoic acid; DKO, double knockout; FXR, farnesoid X receptor; HNF4α, hepatocyte nuclear factor-4α; LBP, l-bifunctional protein; PPARα, peroxisome proliferator receptor α; THCA, trihydroxycholestanoic acid; THC:1-CoA, (24E)-trihydroxycholestenoyl-CoA; VLCS, very long-chain acyl-CoA synthetase; HPLC, high pressure liquid chromatography; MOPS, 4-morpholinepropanesulfonic acid; Pn, postnatal day n; MES, 4-morpholineethanesulfonic acid; THC, trihydroxycholestanoyl-CoA. deficiency. DBP, alternatively called multifunctional protein 2, is involved in the second and third step of the β-oxidation of 2-methyl branched-chain fatty acids or fatty acid derivatives like the C27-bile acid intermediates di- and trihydroxycholestanoic acid (DHCA and THCA) (2Wanders R.J.A. Barth P.G. Heymans H.S.A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Molecular and Metabolic Bases of Inherited Disease. McGraw-Hill, New York2001: 3219-3256Google Scholar). After cleavage of the side chain, the primary C24-bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) are formed. In mice, CDCA is only a minor bile acid, since most of it is converted via hydroxylation into α/β/ω-muri-CA. After synthesis, bile acids are conjugated with taurine or glycine by the bile acyl-CoA:amino acid N-acyltransferase (BAAT) (1Russell D.W. Annu. Rev. Biochem. 2003; 72: 137-174Crossref PubMed Scopus (1406) Google Scholar). In mice, almost exclusively taurine-conjugates are present, because murine BAAT does not use glycine as substrate (3Falany C.N. Fortinberry H. Leiter E.H. Barnes S. J. Lipid Res. 1997; 38: 1139-1148Abstract Full Text PDF PubMed Google Scholar). Patients with a deficiency of DBP accumulate C27-bile acid intermediates, but there are still mature C24-bile acids present in their plasma and bile (4Clayton P.T. Lake B.D. Hjelm M. Stephenson J.B. Besley G.T. Wanders R.J. Schram A.W. Tager J.M. Schutgens R.B. Lawson A.M. J. Inherit. Metab. Dis. 1988; 11: 165-168Crossref PubMed Scopus (38) Google Scholar, 5Une M. Konishi M. Suzuki Y. Akaboshi S. Yoshii M. Kuramoto T. Fujimura K. J. Biochem. (Tokyo). 1997; 122: 655-658Crossref PubMed Scopus (18) Google Scholar). This suggests that an alternative pathway for bile acid biosynthesis exists, which does not involve DBP. Indeed, there is an alternative microsomal pathway for cleavage of the side chain that does not involve any peroxisomal enzymes. This 25-hydroxylation pathway accounts for less than 5% of bile acid synthesis in several species studied (6Duane W.C. Bjorkhem I. Hamilton J.N. Mueller S.M. Hepatology. 1988; 8: 613-618Crossref PubMed Scopus (18) Google Scholar, 7Duane W.C. Pooler P.A. Hamilton J.N. J. Clin. Invest. 1988; 82: 82-85Crossref PubMed Scopus (32) Google Scholar, 8Honda A. Salen G. Shefer S. Matsuzaki Y. Xu G. Batta A.K. Tint G.S. Tanaka N. J. Lipid Res. 2000; 41: 442-451Abstract Full Text Full Text PDF PubMed Google Scholar). Recently, another alternative pathway has been proposed involving the other peroxisomal bifunctional enzyme, LBP (also called multifunctional protein 1), and α-methylacyl-CoA racemase (AMACR) (Fig. 1) (9Cuebas D.A. Phillips C. Schmitz W. Conzelmann E. Novikov D.K. Biochem. J. 2002; 363: 801-807Crossref PubMed Scopus (35) Google Scholar). During peroxisomal β-oxidation of THC-CoA, first (24E)-trihydroxycholestenoyl-CoA (THC:1-CoA) is formed by the branched-chain acyl-CoA oxidase in humans and by trihydroxycholestanoyl-CoA oxidase (ACOX2) in mice. THC:1-CoA is then converted to (24R,25R)-24OH-THC-CoA by the hydratase part of DBP, and subsequently this is converted into 24-keto-THC-CoA by the dehydrogenase part of DBP. Finally, this is thiolytically cleaved by sterol carrier protein X (10Wanders R.J. Vreken P. Ferdinandusse S. Jansen G.A. Waterham H.R. Van Roermund C.W. Van Grunsven E.G. Biochem. Soc. Trans. 2001; 29: 250-267Crossref PubMed Scopus (0) Google Scholar). Alternatively, THC:1-CoA can be converted to (24S,25S)-24OH-THC-CoA by the hydratase part of LBP; however, this is not substrate for the dehydrogenase part of LBP or DBP. In vitro studies have shown that (24S,25S)-24OH-THC-CoA is a substrate for AMACR, which can convert this isomer to the (24S,25R)-isomer, which in its turn can be handled by the dehydrogenase part of LBP (9Cuebas D.A. Phillips C. Schmitz W. Conzelmann E. Novikov D.K. Biochem. J. 2002; 363: 801-807Crossref PubMed Scopus (35) Google Scholar, 11Xu R. Cuebas D.A. Biochem. Biophys. Res. Commun. 1996; 221: 271-278Crossref PubMed Scopus (36) Google Scholar). If this route would be operational in vivo, it could account for some residual bile acid synthesis in DBP-deficient patients. In addition, the (24S)-hydroxycholesterol, which is formed in order to eliminate cholesterol from the brain and is excreted across the blood-brain barrier into the circulation, can be converted into bile acids, after ω-oxidation, via LBP (12Bjorkhem I. Andersson U. Ellis E. Alvelius G. Ellegard L. Diczfalusy U. Sjovall J. Einarsson C. J. Biol. Chem. 2001; 276: 37004-37010Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). (24S,25S)-24OH-THC-CoA is present in body fluids of patients with DBP deficiency, indicating that in these patients LBP indeed forms this hydroxy-intermediate (13Vreken P. van Rooij A. Denis S. van Grunsven E.G. Cuebas D.A. Wanders R.J. J. Lipid Res. 1998; 39: 2452-2458Abstract Full Text Full Text PDF PubMed Google Scholar). The question remains, however, whether the alternative pathway via LBP really contributes to bile acid synthesis when DBP functions normally or whether it can partly take over the role of DBP in bile acid synthesis in case of a deficiency of DBP. To answer this question, we performed bile acid analysis in LBP, DBP, and LBP:DBP double knock-out (DKO) mice. DBP–/– mice exhibit a severe postnatal growth retardation and a variable life span, with more than 50% of the mice dying before weaning, whereas the others survive into adulthood. In adult DBP–/– mice, C27-bile acid intermediates have been identified, as expected, but C24-bile acids are still present (14Baes M. Huyghe S. Carmeliet P. Declercq P.E. Collen D. Mannaerts G.P. Van Veldhoven P.P. J. Biol. Chem. 2000; 275: 16329-16336Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Although the creation of the LBP–/– mouse has been published in 1996 and no gross phenotypic defects were described (15Qi C. Zhu Y. Pan J. Usuda N. Maeda N. Yeldandi A.V. Rao M.S. Hashimoto T. Reddy J.K. J. Biol. Chem. 1999; 274: 15775-15780Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), no specific bile acid analysis in this mouse model has been reported. LBP:DBP DKO mice have a complete block at the level of the second step of peroxisomal β-oxidation. They exhibit severe growth retardation and postnatal mortality, with almost none surviving beyond weaning (16Jia Y. Qi C. Zhang Z. Hashimoto T. Rao M.S. Huyghe S. Suzuki Y. Van Veldhoven P.P. Baes M. Reddy J.K. J. Biol. Chem. 2003; 278: 47232-47239Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). It is unclear why the DKO mice are much more severely affected than the single DBP knock-out mice, because at this moment no specific function has been attributed to LBP. In order to compare bile acid composition, we collected liver, plasma, and bile of mice with the different genotypes between postnatal day 2 (P2) and adulthood. Because of the poor survival of the DKO mice, we could only analyze samples of these mice until P14–15. Since we noticed a major difference in the conjugation of bile acids in young animals compared with adult animals, a second goal was to investigate the ontogeny of hepatic bile acid conjugation. Animals—Wild type and single DBP knock-out (originally named multifunctional protein 2 knock-out) mice were obtained by inbreeding LBP:DBP+/+:+/– mice, whereas LBP knock-out and DKO mice were descendants of LBP:DBP–/–:+/– breeding pairs (14Baes M. Huyghe S. Carmeliet P. Declercq P.E. Collen D. Mannaerts G.P. Van Veldhoven P.P. J. Biol. Chem. 2000; 275: 16329-16336Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 15Qi C. Zhu Y. Pan J. Usuda N. Maeda N. Yeldandi A.V. Rao M.S. Hashimoto T. Reddy J.K. J. Biol. Chem. 1999; 274: 15775-15780Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 17Baes M. Gressens P. Huyghe S. De N.K. Qi C. Jia Y. Mannaerts G.P. Evrard P. Van V.P. Declercq P.E. Reddy J.K. J. Neuropathol. Exp. Neurol. 2002; 61: 368-374Crossref PubMed Scopus (43) Google Scholar). Mice were bred in the animal housing facility of the University of Leuven under conventional conditions. They had unlimited access to standard rodent chow (Muracon-G; Carfil Quality-Pavan Services, Oud-Turn-hout, Belgium; for breeding pairs, this was enriched in a ratio of 1:1 with AM-II (Hope Farms, Arie Blok, Woerden, The Netherlands)) and water and were kept on a 12-h light/dark cycle. All animal experiments were approved by the Institutional Animal Ethical Committee of the University of Leuven. Wild type and peroxisome proliferator receptor α (PPARα)–/– mice on a Sv/129 genetic background, which were used for bile acyl-CoA synthetase and bile acyl-CoA:amino acid acyltransferase activity measurements, were obtained from the Jackson Laboratory. Bile Acid Analysis—Bile acids were analyzed by HPLC-negative ion electrospray tandem mass spectrometry as described (18Bootsma A.H. Overmars H. van Rooij A. van Lint A.E. Wanders R.J. van Gennip A.H. Vreken P. J. Inherit. Metab. Dis. 1999; 22: 307-310Crossref PubMed Scopus (65) Google Scholar) with only minor modifications. The HPLC column used was a C8-column (Phenomenex Luna; 50 × 1 mm), and the bile acids were separated using a gradient from 60:40 (ammonium formiate, pH 8.1/MeOH (v/v)) to 90:10 (acetonitrile/H2O (v/v)) at a flow rate of 60 μl/min. Sample preparation for plasma was performed as described in Ref. 18Bootsma A.H. Overmars H. van Rooij A. van Lint A.E. Wanders R.J. van Gennip A.H. Vreken P. J. Inherit. Metab. Dis. 1999; 22: 307-310Crossref PubMed Scopus (65) Google Scholar. For bile acid analysis in bile, the bile was diluted 500 times in H2O. For analysis in liver, 15–25 mg of liver (wet weight) was homogenized in 150 μl of H2O. After sonication, 150 μl of MeOH was added plus 100 μl of internal standard ([2,2,4,4-2H]tauro-CA, [2,2,4,4-2H4]tauro-CDCA, [2,2,4,4-2H4]glyco-CA, [2,2,4,4-2H4]glyco-CDCA, [2,2,4,4-2H4]CA, and [2,2,4,4-2H4]CDCA). After another round of sonication, the sample was deproteinized by the addition of 750 μl of acetonitrile followed by subsequent centrifugation for 10 min at 20,000 × g at 4 °C. The supernatant was removed and stored briefly, whereas the pellet was redissolved in 300 μl of MeOH/H2O (1:1), sonicated, and deproteinized as described above. The supernatants were pooled and evaporated under a stream of N2, and the residue was dissolved in 250 μl of MeOH/H2O (1:2). Five μl was injected into the HPLC tandem mass spectrometric system. Quantitation of the various conjugated bile acids was done using multiple reaction monitoring, whereas the various unconjugated bile acids were detected with selected ion monitoring. Peroxisomal Acyl-CoA Oxidase Measurements—Acyl-CoA oxidase activity measurements were performed essentially as described before (19Wanders R.J. Denis S.W. Dacremont G. J. Biochem. (Tokyo). 1993; 113: 577-582Crossref PubMed Scopus (18) Google Scholar). H2O2 production by the action of the acyl-CoA oxidases was measured in mouse liver homogenates prepared in PBS with 25 μm FAD by fluorometric quantitation of H2O2 using homovanillic acid and horseradish peroxidase. Measurements were carried out in the following incubation mixture: 50 mm MOPS-NaOH (pH 7.6), 1 mm homovanillic acid, 18 units/ml horseradish peroxidase, 0.1 mm NaN3,5 μm FAD, and 5 μm bovine serum albumin. The reactions were started by the addition of 50 μm C16-CoA, 50 μm pristanoyl-CoA, or 80 μm THC-CoA. Fluorescence was followed at 30-s intervals for 10 min using a Cobas Bio centrifugal analyzer (excitation wavelength, 327 nm; emission filter, 410–490 nm) (Hoffman-La Roche). Bile Acyl-CoA Synthetase Activity Measurements—Bile acid synthetase activity was measured with CA (sodium salt; Merck; stock 1 mm in H2O) and THCA (Ten Brink, Amsterdam, The Netherlands; stock 2 mm in 10 mg/ml β-cyclodextrin, 100 mm Tris, pH 8) as substrate. The incubations consisted of 100 mm Tris (pH 8), 10 mm ATP, 10 mm MgCl2, 1mm CoA, 0.5 mm dithiothreitol, 20 μm bovine serum albumin, and 100 μm CA or THCA in a final volume of 100 μl. The reactions were started by the addition of mouse liver homogenate (0.025 mg/ml) prepared in PBS and terminated after 30 min at 37 °C by adding 10 μl of 2 m HCl. Subsequently, the incubation mixtures were neutralized with 12 μl of MES/KOH (0.6 m/2 m) and deproteinized by adding 50 μl of acetonitrile. After centrifugation for 10 min at 20,000 × g at 4 °C, the supernatants were applied to a reverse-phase C18-column (Alltima, 250 × 4.6 mm; Alltech). Resolution of the different CoA esters was achieved by elution with a linear gradient of acetonitrile (25–37% (v/v)) in 16.9 mm sodium phosphate buffer (pH 6.9) under continuous monitoring of the absorbance at 254 nm. Bile Acyl-CoA:Amino Acid Acyltransferase Activity Measurements— Bile acyl-CoA:amino acid acyltransferase activity was measured with taurine and CA-CoA (chemically synthesized as described (20Rasmussen J.T. Borchers T. Knudsen J. Biochem. J. 1990; 265: 849-855Crossref PubMed Scopus (161) Google Scholar)) as substrates. The incubations consisted of 100 mm potassium phosphate buffer (pH 8), 20 mm taurine, 10 mm ATP, 10 mm MgCl2,1mm CoA, 0.5 mm dithiothreitol, 20 μm bovine serum albumin, and 200 μm CA-CoA in a final volume of 100 μl. The reactions were started by the addition of mouse liver homogenate (0.2 mg/ml) prepared in PBS and terminated after 30 min at 37 °C by the addition of 500 μl of acetonitrile. Fifty μlof [2,2,4,4-2H4]tauro-CA and [2,2,4,4-2H4]CA was added as an internal standard, and the sample was centrifuged for 10 min at 20,000 × g at 4 °C. The supernatant was evaporated under a stream of N2, and the residue was dissolved in 100 μl of MeOH/H2O (1:2). Five μl was injected into the HPLC tandem mass spectrometric system. Subcellular fractions were prepared from the liver of overnight fasted mice homogenized in 0.25 m sucrose, 5 mm MOPS, pH 7.2, 1 mm EDTA, and 0.1% ethanol (21van Veldhoven P.P. Just W.W. Mannaerts G.P. J. Biol. Chem. 1987; 262: 4310-4318Abstract Full Text PDF PubMed Google Scholar) and analyzed for protein content, catalase (peroxisomal marker), lactate dehydrogenase (cytosolic marker) (21van Veldhoven P.P. Just W.W. Mannaerts G.P. J. Biol. Chem. 1987; 262: 4310-4318Abstract Full Text PDF PubMed Google Scholar), and BAAT activity as described above. Northern Blotting—RNA was extracted from liver using the TRIzol® reagent (Invitrogen) and analyzed by Northern blotting as previously described (22Huyghe S. Casteels M. Janssen A. Meulders L. Mannaerts G.P. Declercq P.E. Van Veldhoven P.P. Baes M. Biochem. J. 2001; 353: 673-680Crossref PubMed Scopus (44) Google Scholar). The blots were consecutively hybridized with radioactive probes and exposed. The probes were generated by reverse transcription-PCR on mouse liver RNA using the following primers: mBAAT (5′-GCCAAGCTGACAGCTGTTC-3′ and 5′-AGGAGATGCCCAGCTCC-3′, based on BC012683), VLCS (5′-TGCCAGTGCTCTACACCG-3′ and 5′-TGCGAGGTCTATCGAGTTTC-3′, based on AF033031) (renamed to SLC27A2), mBACS (5′-GGGTATTTGGAAGAAACTAAC-3′ and 5′-GGACAACTTTGTGAAGCTG-3′, based on BC013272) (renamed to SLC27A5), mFXR (5′-ATGGTGATGCAGTTTCAGGGCTTAG-3′ and 5′-TCACTGCACATCCCAGATCTCACAG-3′, based on NM009108), and mβ-actin (5′-GCATTGTTACCAACTGGG-ACGACATGG-3′ and 5′-CTTCATGGTGCTAGG-3′, based on NM007393). Western Blotting—Western blotting was performed with antibodies against farnesoid X receptor (FXR), hepatocyte nuclear factor-4α (HNF4α), and calregulin. Mouse liver homogenate (50 μg in case of FXR and HNF4α, 25 μg for calregulin) was subjected to electrophoresis on a 10% (w/v) SDS-polyacrylamide gel essentially as described by Laemmli (23Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar) and transferred to a nitrocellulose sheet. After blocking of nonspecific binding sites with 50 g/liter Profitar and 10 g/liter bovine serum albumin in 1 g/liter Tween 20/PBS for 1 h, the blot was incubated for 2 h with one of the following primary antibodies: anti-FXR (diluted 1:200 in 3 g/liter bovine serum albumin; Santa Cruz Biotechnology, Santa Cruz, CA), anti-HNF4α (diluted 1:100; Perseus Proteomics Inc., Tokyo, Japan), or anti-calregulin (diluted 1:1000; Santa Cruz Biotechnology). Goat anti-rabbit (FXR and calregulin) or goat anti-mouse (HNF4α) IgG antibodies conjugated to alkaline phosphatase were used for detection, according to the manufacturer's instructions (Bio-Rad). Statistical Analyses—Data are expressed as mean ± S.D. Statistical significance was evaluated using an unpaired Student's t test. The results were considered significant when p ≤ 0.05. Bile Acid Analysis—Bile acids were analyzed in bile, liver, and plasma from LBP, DBP, and LBP:DBP DKO mice. Since almost no DKO mice survive beyond weaning, comparative analysis in mice from all the different genotypes could only be performed before postnatal day 21 (P21). Samples from the single knock-out mice were also analyzed at an adult age. In bile, liver, and plasma from wild type and LBP–/– mice (age <P21) the major bile acids were tauro-CA, tauro-muri-CA, and tauro-OH-CA. The mass spectra of bile from LBP–/– mice were similar to those from wild type mice (see Fig. 2), revealing only very limited amounts of bile acid biosynthesis intermediates. In contrast, C27-bile acid intermediates were the major bile acids in bile, liver, and plasma from both DBP–/– and DKO mice. DBP–/– mice accumulated mainly THCA with one double bond (THC:1), which has previously been identified by mass spectrometric analysis as the first intermediate of the peroxisomal β-oxidation process (14Baes M. Huyghe S. Carmeliet P. Declercq P.E. Collen D. Mannaerts G.P. Van Veldhoven P.P. J. Biol. Chem. 2000; 275: 16329-16336Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). DKO mice, however, accumulated predominantly THCA itself or OH-THCA. The exact position of the hydroxyl group could not be identified, but the accumulating OH-THCA is most likely a mixture of THCA hydroxylated at several different positions, because multiple peaks were observed by HPLC for the molecule with m/z 465. In liver of both DBP–/– and DKO mice, the accumulation of C27-bile acid intermediates was accompanied by a severe deficiency of C24-bile acids (see Fig. 3). At P2–4 the liver of wild type animals contained 362 ± 88 nmol/g (wet weight) C24-bile acids, whereas the sum of all C24-bile acids in liver from DBP–/– mice was 34 ± 2 nmol/g, and in liver from the DKO mice 25 ± 10 nmol/g. This resulted in a C27/C24 ratio of 2.6 in DBP–/– mice and 5.1 in DKO mice versus a ratio of 0.01 in wild type mice. The total amount of bile acids (C24 + C27) was also reduced in DBP–/– and DKO mouse liver (in wild type animals 367 ± 87, in DBP–/– mice 121 ± 18, and in DKO mice 122 ± 30 nmol/g (w/w); see Fig. 3). At P10–11, the deficiency of C24-bile acids was even more pronounced in DKO mice (10 ± 3 nmol/g), resulting in a C27/C24 ratio of 13.3 in DKO mice versus 1.9 in DBP–/– mice. Surprisingly, at this age, livers from LBP–/– mice also contained reduced levels of C24-bile acids (243 ± 17 nmol/g versus 325 ± 80 nmol/g in wild type mice). In contrast to the DBP–/– and DKO mice, there was no gross accumulation of C27-bile acid intermediates in LBP–/– livers (6.2 ± 2.7 nmol/g in LBP–/– mice versus 3.2 ± 0.7 nmol/g in wild type mice, 84 ± 23 nmol/g in DBP–/– mice, and 130 ± 57 nmol/g in DKO mice). A comparative analysis of bile acid content was performed in liver from eight LBP:DBP–/–:+/+ and seven LBP:DBP–/–: +/– mice (P10–11) to study whether subtle changes between mice of these two genotypes could help clarify the role of LBP in bile acid biosynthesis. However, no differences in bile acid levels or patterns were found (see Table I). At P2–4 and in 6–10-week-old LBP–/– mice, the amount of C24-bile acids was completely normal. This was not the case in liver of DBP–/– mice, where C24-bile acids remained very low at all ages studied (P10–11, P21, 6–10 weeks).Table IBile acid levels in liver from LBP:DBP–/–:+/+ and LBP:DBP –/–:+/– mice (P10–11)Total C24-bile acidsTotal C27-bile acidsTotal bile acidsnmol/gLBP:DBP-/-:+/+ (n = 8)an = number. All measurements were performed in duplicate.233 ± 925.9 ± 3.5239 ± 94LBP:DBP-/-:+/- (n = 7)256 ± 466.6 ± 1.5262 ± 46a n = number. All measurements were performed in duplicate. Open table in a new tab Although the concentration of bile acids in plasma is low, the situation in the liver is relatively well reflected in plasma (Fig. 4). DBP–/– and DKO mice accumulate C27-bile acids, whereas they have a deficiency of C24-bile acids. This deficiency is most pronounced before weaning, but also at an adult age DBP–/– mice have statistically lower levels of C24-bile acids in their plasma. At P7–9, the sum of all C24-bile acids in plasma from DBP–/– mice is 3.5% from the C24-bile acids present in plasma from wild type animals. At P14–15, this is 7.7%; at P21 it is 27.6%; and at the age of 6–10 weeks it is 41.2%. This increasing percentage is not only due to a small increase in C24-bile acids of about 1.3-fold in DBP–/– mice but is mostly because of a strong decrease of bile acids in plasma from wild type animals (and also LBP–/– mice). Age-dependent changes in bile acid concentrations were even more pronounced in bile of DBP–/– mice (Fig. 5). C24-bile acids gradually increased with age, equaling levels in wild type mice at 5–6 months. At this age, bile from DBP–/– mice could only be distinguished from wild type and LBP–/– bile because of the presence of C27-bile acids. Together with a simultaneous age-dependent increase of C27-bile acids in bile of DBP–/– mice, this gave rise to elevated total bile acid levels in bile of 5–6-month-old DBP–/– mice. At all ages, the ratio of C27/C24-bile acids in bile of DBP–/– mice was much lower than in plasma and liver (at 6–10 weeks of age, the ratio was 1.2 in bile compared with 3.5 and 9.0 in liver and plasma, respectively).Fig. 5Bile acids measured in bile from wild type, LBP–/–, DBP–/–, and LBP:DBP–/–:–/– mice during development from the neonatal period until adulthood. Total C24-bile acids is the sum of t-(m)CA and (m)CA; total C27-bile acids is the sum of t-THCA, t-THC:1, OH-THCA, OH-THC:1, THCA, and THC:1; total bile acids is the sum of all C24- and C27-bile acids described above; the taurine/free C27 ratio is the ratio of the taurine-conjugated C27-bile acids over the free C27-bile acids described. The number of animals per group has been indicated in or just above the bars. *, p ≤ 0.05; **, p ≤ 0.005.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A surprising observation was that the accumulating C27-bile acids in DBP–/– mice were predominantly unconjugated before weaning in liver, bile, and plasma, whereas they were mostly present as taurine-conjugates at adult age (the ratio of taurine/free C27-bile acids in liver of DBP–/– mice at P2–4 was 0.13 ± 0.06; at P10–11, 0.53 ± 0.34; at P21, 1.34 ± 0.63; and 3.37 ± 1.03 in 6–10-week-old animals; see Figs. 3, 4, 5). This age-dependent increase in conjugation of C27-bile acid intermediates was in sharp contrast to the situation with the C24-bile acids, which were already fully conjugated at P2–4 both in wild type and DBP–/– mice. Even in adult DBP–/– mice, about 23% of the C27-bile acids were unconjugated, whereas only 5% of the C24-bile acids were unconjugated in the same animals. Peroxisomal Acyl-CoA Oxidase Measurements—Because, in contrast to our expectation, DKO mice accumulated mainly THCA and hydroxylated THCA instead of THC:1, we measured peroxisomal acyl-CoA oxidase activities in liver from wild type, LBP–/–, DBP–/–, and DKO mice at P10–11 with THC-CoA, pristanoyl-CoA, and, for comparison, with C16-CoA (see Table II). The oxidation rate of pristanoyl-CoA and THC-CoA was markedly increased in DBP–/– as compared with wild type livers, which is in agreement with earlier results demonstrating increased protein levels for ACOX2 (14Baes M. Huyghe S. Carmeliet P. Declercq P.E. Collen D. Mannaerts G.P. Van Veldhoven P.P. J. Biol. Chem. 2000; 275: 16329-16336Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). In contrast, in DKO mice, acyl-CoA oxidase activity measured with pristanoyl-CoA was strongly reduced and was even not detectable with THC-CoA as substrate. The residual conversion of pristanoyl-CoA could very well be due to the activity of ACOX3. The lack of THC-CoA oxidase activity explains the absence of THC:1 in liver, bile, and plasma from DKO mice. Straight-chai" @default.
• W2088794057 created "2016-06-24" @default.
• W2088794057 creator A5009514619 @default.
• W2088794057 creator A5021119790 @default.
• W2088794057 creator A5035150830 @default.
• W2088794057 creator A5040743356 @default.
• W2088794057 creator A5045855976 @default.
• W2088794057 creator A5072933549 @default.
• W2088794057 creator A5084691397 @default.
• W2088794057 creator A5087611764 @default.
• W2088794057 date "2005-05-01" @default.
• W2088794057 modified "2023-09-26" @default.
• W2088794057 title "Developmental Changes of Bile Acid Composition and Conjugation in L- and D-Bifunctional Protein Single and Double Knockout Mice" @default.
• W2088794057 cites W124149417 @default.
• W2088794057 cites W1556317573 @default.
• W2088794057 cites W1562127751 @default.
• W2088794057 cites W1675820479 @default.
• W2088794057 cites W1918619761 @default.
• W2088794057 cites W1970340553 @default.
• W2088794057 cites W1973826150 @default.
• W2088794057 cites W1983212413 @default.
• W2088794057 cites W1991975419 @default.
• W2088794057 cites W2011569902 @default.
• W2088794057 cites W2014879410 @default.
• W2088794057 cites W2050777257 @default.
• W2088794057 cites W2051559963 @default.
• W2088794057 cites W2056396219 @default.
• W2088794057 cites W2075464063 @default.
• W2088794057 cites W2084011989 @default.
• W2088794057 cites W2087563317 @default.
• W2088794057 cites W2088633754 @default.
• W2088794057 cites W2089450781 @default.
• W2088794057 cites W2100837269 @default.
• W2088794057 cites W2101355612 @default.
• W2088794057 cites W2106515170 @default.
• W2088794057 cites W2132291792 @default.
• W2088794057 cites W2132815603 @default.
• W2088794057 cites W2134778685 @default.
• W2088794057 cites W2136087941 @default.
• W2088794057 cites W2148022169 @default.
• W2088794057 cites W2170486795 @default.
• W2088794057 cites W2170615733 @default.
• W2088794057 cites W2172210205 @default.
• W2088794057 cites W2336414129 @default.
• W2088794057 cites W2336686047 @default.
• W2088794057 cites W4231809167 @default.
• W2088794057 cites W4250505098 @default.
• W2088794057 cites W4251451246 @default.
• W2088794057 cites W4255314005 @default.
• W2088794057 doi "https://doi.org/10.1074/jbc.m414311200" @default.
• W2088794057 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15769750" @default.
• W2088794057 hasPublicationYear "2005" @default.
• W2088794057 type Work @default.
• W2088794057 sameAs 2088794057 @default.
• W2088794057 citedByCount "44" @default.
• W2088794057 countsByYear W20887940572012 @default.
• W2088794057 countsByYear W20887940572013 @default.
• W2088794057 countsByYear W20887940572014 @default.
• W2088794057 countsByYear W20887940572016 @default.
• W2088794057 countsByYear W20887940572018 @default.
• W2088794057 countsByYear W20887940572019 @default.
• W2088794057 countsByYear W20887940572020 @default.
• W2088794057 countsByYear W20887940572021 @default.
• W2088794057 countsByYear W20887940572022 @default.
• W2088794057 countsByYear W20887940572023 @default.
• W2088794057 crossrefType "journal-article" @default.
• W2088794057 hasAuthorship W2088794057A5009514619 @default.
• W2088794057 hasAuthorship W2088794057A5021119790 @default.
• W2088794057 hasAuthorship W2088794057A5035150830 @default.
• W2088794057 hasAuthorship W2088794057A5040743356 @default.
• W2088794057 hasAuthorship W2088794057A5045855976 @default.
• W2088794057 hasAuthorship W2088794057A5072933549 @default.
• W2088794057 hasAuthorship W2088794057A5084691397 @default.
• W2088794057 hasAuthorship W2088794057A5087611764 @default.
• W2088794057 hasBestOaLocation W20887940571 @default.
• W2088794057 hasConcept C138885662 @default.
• W2088794057 hasConcept C161790260 @default.
• W2088794057 hasConcept C185592680 @default.
• W2088794057 hasConcept C2779399885 @default.
• W2088794057 hasConcept C2780443040 @default.
• W2088794057 hasConcept C40231798 @default.
• W2088794057 hasConcept C41895202 @default.
• W2088794057 hasConcept C55493867 @default.
• W2088794057 hasConceptScore W2088794057C138885662 @default.
• W2088794057 hasConceptScore W2088794057C161790260 @default.
• W2088794057 hasConceptScore W2088794057C185592680 @default.
• W2088794057 hasConceptScore W2088794057C2779399885 @default.
• W2088794057 hasConceptScore W2088794057C2780443040 @default.
• W2088794057 hasConceptScore W2088794057C40231798 @default.
• W2088794057 hasConceptScore W2088794057C41895202 @default.
• W2088794057 hasConceptScore W2088794057C55493867 @default.
• W2088794057 hasIssue "19" @default.
• W2088794057 hasLocation W20887940571 @default.
• W2088794057 hasOpenAccess W2088794057 @default.
• W2088794057 hasPrimaryLocation W20887940571 @default.
• W2088794057 hasRelatedWork W2065583757 @default.
• W2088794057 hasRelatedWork W2302922649 @default.
• W2088794057 hasRelatedWork W2343617961 @default.

• next