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W2065578656 abstract "Sterol 27-hydroxylase is important for the degradation of the steroid side chain in conversion of cholesterol into bile acids and has been ascribed a regulatory role in cholesterol homeostasis. Its deficiency causes the autosomal recessive disease cerebrotendinous xanthomatosis (CTX), characterized by progressive dementia, xanthomatosis, and accelerated atherosclerosis.Mice with a disrupted cyp27 (cyp27 −/−) had normal plasma levels of cholesterol, retinol, tocopherol, and 1,25-dihydroxyvitamin D. Excretion of fecal bile acids was decreased (<20% of normal), and formation of bile acids from tritium-labeled 7α-hydroxycholesterol was less than 15% of normal. Compensatory up-regulation of hepatic cholesterol 7α-hydroxylase and hydroxymethylglutaryl-CoA reductase (9- and 2–3-fold increases in mRNA levels, respectively) was found. No CTX-related pathological abnormalities were observed. In CTX, there is an increased formation of 25-hydroxylated bile alcohols and cholestanol. In bile and feces of thecyp27 −/− mice only traces of bile alcohols were found, and there was no cholestanol accumulation.It is evident that sterol 27-hydroxylase is more important for bile acid synthesis in mice than in humans. The results do not support the contention that 27-hydroxylated steroids are critical for maintenance of cholesterol homeostasis or levels of vitamin D metabolites in the circulation. Sterol 27-hydroxylase is important for the degradation of the steroid side chain in conversion of cholesterol into bile acids and has been ascribed a regulatory role in cholesterol homeostasis. Its deficiency causes the autosomal recessive disease cerebrotendinous xanthomatosis (CTX), characterized by progressive dementia, xanthomatosis, and accelerated atherosclerosis. Mice with a disrupted cyp27 (cyp27 −/−) had normal plasma levels of cholesterol, retinol, tocopherol, and 1,25-dihydroxyvitamin D. Excretion of fecal bile acids was decreased (<20% of normal), and formation of bile acids from tritium-labeled 7α-hydroxycholesterol was less than 15% of normal. Compensatory up-regulation of hepatic cholesterol 7α-hydroxylase and hydroxymethylglutaryl-CoA reductase (9- and 2–3-fold increases in mRNA levels, respectively) was found. No CTX-related pathological abnormalities were observed. In CTX, there is an increased formation of 25-hydroxylated bile alcohols and cholestanol. In bile and feces of thecyp27 −/− mice only traces of bile alcohols were found, and there was no cholestanol accumulation. It is evident that sterol 27-hydroxylase is more important for bile acid synthesis in mice than in humans. The results do not support the contention that 27-hydroxylated steroids are critical for maintenance of cholesterol homeostasis or levels of vitamin D metabolites in the circulation. Sterol 27-hydroxylase is a mitochondrial species of cytochrome P-450 with a broad tissue and organ distribution and with a broad substrate specificity (for a review, see Ref. 1Björkhem I. J. Lipid Res. 1992; 33: 455-471Abstract Full Text PDF PubMed Google Scholar). The enzyme is important for bile acid biosynthesis but has also been ascribed a role in connection with cholesterol removal from extrahepatic tissues, in regulation of cholesterol homeostasis, and in metabolism of vitamin D. Sterol 27-hydroxylase is responsible for the first step in the degradation of the steroid side chain in connection with bile acid biosynthesis in the liver. In the major pathway to bile acids in mammalian liver, 7α-hydroxylation of cholesterol is the first and rate-limiting step, and 27-hydroxylation occurs at a later stage with a 7α-hydroxylated intermediate as substrate. In an alternative pathway, 27-hydroxylation of cholesterol is the first step in the sequence, followed by a 7α-hydroxylation catalyzed by a specific oxysterol 7α-hydroxylase (1Björkhem I. J. Lipid Res. 1992; 33: 455-471Abstract Full Text PDF PubMed Google Scholar). In a recently discovered minor pathway, extrahepatic sterol 27-hydroxylase converts cholesterol into 27-hydroxycholesterol or 3β-hydroxy-5-cholestenoic acid (2Björkhem I. Andersson O. Diczflusy U. Sevastik B. Xiu R.J. Duan C. Lund E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8592-8596Crossref PubMed Scopus (283) Google Scholar, 3Lund E. Andersson O. Zhang J. Babiker A. Ahlborg G. Diczfalusy U. Einarsson K. Sjövall J. Björkhem I. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 208-212Crossref PubMed Scopus (147) Google Scholar). The latter compounds are transported to the liver and converted into bile acids. Sterol 27-hydroxylase has also 25-hydroxylase activity toward vitamin D (4Ohyama Y. Masumoto O. Usui E. Okuda K. J. Biochem. (Tokyo). 1991; 109: 389-393Crossref PubMed Scopus (27) Google Scholar) and 1α-hydroxylase activity toward 25-hydroxyvitamin D (5Axén E. Postlind H. Sjöberg H. Wikvall K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10014-10018Crossref PubMed Scopus (68) Google Scholar). Since there is also a microsomal cytochrome P-450 that catalyzes 25-hydroxylation of vitamin D (6Björkhem I. Hansson R. Holmberg I. Wikvall K. Biochem. Biophys. Res. Commun. 1979; 90: 615-622Crossref PubMed Scopus (21) Google Scholar), the relative importance of the sterol 27-hydroxylase is not known. 27-Hydroxycholesterol, formed from cholesterol by the sterol 27-hydroxylase, is a potent down-regulator of cholesterol synthesis in cultured cells (for reviews see Refs. 7Javitt N.B. J. Lipid Res. 1990; 31: 1527-1533Abstract Full Text PDF PubMed Google Scholar and 8Lund E. Björkhem I. Acc. Chem. Res. 1995; 28: 241-249Crossref Scopus (51) Google Scholar). On the basis of this, and on the basis of studies with sterol 27-hydroxylase inhibitors, sterol 27-hydroxylase has been suggested to have a regulatory role in cholesterol homeostasis. The relative importance of this mechanism is controversial, however. Based on studies with cholesterol specifically deuterium-labeled in the 27-position, which retards the rate of 27-hydroxylation, we showed that sterol 27-hydroxylase activity is of little or no direct importance for cholesterol-induced suppression of cholesterol synthesis in mouse liver (9Lund E. Breuer O. Björkhem I. J. Biol. Chem. 1992; 267: 25092-25097Abstract Full Text PDF PubMed Google Scholar). It is now well documented that patients with the rare disease cerebrotendinous xanthomatosis (CTX) 1The abbreviations used are: CTX, cerebrotendinous xanthomatosis; PCR, polymerase chain reaction; bp, base pair; kb, kilobase pair; HMG-CoA, hydroxymethylglutaryl-CoA. have a deficiency of sterol 27-hydroxylase (for a review, see Ref. 10Björkhem I. Muri-Boberg K. Scriver C.R. Beaudet A.L. Sly W.S. Valee D. The Metabolic Basis of Inherited Diseases. McGraw-Hill Inc., New York1994: 2073-2100Google Scholar). Recently, a number of mutations have been defined in the sterol 27-hydroxylase gene of these patients (11Cali J.J. Hsieh C.L. Francke U. Russell D.W. J. Biol. Chem. 1991; 266: 7779-7783Abstract Full Text PDF PubMed Google Scholar, 12Leitersdorf E. Reshef A. Meiner V. Levitzki R. Pressman-Schwartz S. Dann E.J. Berkman N. Cali J.J. Klapholz L. Berginer V.M. J. Clin. Invest. 1993; 91: 2488-2496Crossref PubMed Scopus (155) Google Scholar, 13Meiner V. Marais D.A. Reshef A. Björkhem I. Leitersdorf E. Hum. Mol. Genet. 1994; 3: 193-194Crossref PubMed Scopus (23) Google Scholar, 14Kim K.S. Kubota S. Kuriyama M. Fujiyama J. Björkhem I. Eggertsen G. Seyama Y. J. Lipid Res. 1994; 35: 1031-1039Abstract Full Text PDF PubMed Google Scholar, 15Meiner V. Meiner Z. Reshef A. Björkhem I. Leitersdorf E. Neurology. 1994; 44: 288-290Crossref PubMed Google Scholar, 16Reshef A. Meiner V. Berginer V.M. Leitersdorf E. J. Lipid Res. 1994; 35: 478-483Abstract Full Text PDF PubMed Google Scholar, 17Leitersdorf E. Safadi R. Meiner V. Reshef A. Bjorkhem I. Friedlander Y. Morkos S. Berginer V.M. Am. J. Hum. Genet. 1994; 55: 907-915PubMed Google Scholar, 18Segev H. Reshef A. Clavey V. Delbart C. Routier G. Leitersdorf E. Hum. Mol. Genet. 1995; 95: 238-240Google Scholar, 19Watts G.F. Mitchell W.D. Bending J.J. Reshef A. Leitersdorf E. Q. J. Med. 1996; 89: 55-63Crossref Scopus (32) Google Scholar, 20Nakashima N. Sakai Y. Sakai H. Yanese T. Haji M. Umeda F. Koga S. Hoshita T. Nawata H. J. Lipid Res. 1994; 35: 663-668Abstract Full Text PDF PubMed Google Scholar). As a result of the enzymatic defect, patients with CTX have a reduced synthesis of bile acids, in particular chenodeoxycholic acid (10Björkhem I. Muri-Boberg K. Scriver C.R. Beaudet A.L. Sly W.S. Valee D. The Metabolic Basis of Inherited Diseases. McGraw-Hill Inc., New York1994: 2073-2100Google Scholar). Cholic acid is formed in these patients by a pathway involving 25-hydroxylated bile alcohols as intermediates. Under normal conditions this alternative pathway is of little or no importance, both in rats and man (21Duane W.E. Björkhem I. Hamilton J.N. Mueller S.M. Hepatology. 1988; 8: 613-618Crossref PubMed Scopus (18) Google Scholar, 22Duane W.E. Pooler P.A. Hamilton J.N. J. Clin. Invest. 1988; 82: 82-85Crossref PubMed Scopus (32) Google Scholar). Due to the reduced formation of bile acids, the negative feedback of the cholesterol 7α-hydroxylase is reduced, resulting in an up-regulation of this enzyme. As a consequence, gram amounts of bile alcohols are formed and excreted in bile and feces. Another consequence of the disease is excess formation and accumulation of cholestanol (10Björkhem I. Muri-Boberg K. Scriver C.R. Beaudet A.L. Sly W.S. Valee D. The Metabolic Basis of Inherited Diseases. McGraw-Hill Inc., New York1994: 2073-2100Google Scholar). Most of this formation seems to be secondary to the accumulation of an intermediate in bile acid biosynthesis, 7α-hydroxy-4-cholesten-3-one. This steroid can be converted into cholestanol by hepatic enzymes. Patients with CTX have normal cholesterol levels in the circulation (17Leitersdorf E. Safadi R. Meiner V. Reshef A. Bjorkhem I. Friedlander Y. Morkos S. Berginer V.M. Am. J. Hum. Genet. 1994; 55: 907-915PubMed Google Scholar). Despite this they develop xanthomas and premature atherosclerosis. The possibility has been discussed that this may be due to the reduced elimination of cholesterol from macrophages by the sterol 27-hydroxylase (1Björkhem I. J. Lipid Res. 1992; 33: 455-471Abstract Full Text PDF PubMed Google Scholar, 2Björkhem I. Andersson O. Diczflusy U. Sevastik B. Xiu R.J. Duan C. Lund E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8592-8596Crossref PubMed Scopus (283) Google Scholar, 3Lund E. Andersson O. Zhang J. Babiker A. Ahlborg G. Diczfalusy U. Einarsson K. Sjövall J. Björkhem I. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 208-212Crossref PubMed Scopus (147) Google Scholar). Disturbances in vitamin D metabolism have been described in a few cases of CTX (23Berginer V.M. Salen G. Shefer S. Neurol. Clin. 1989; 7: 55-64Abstract Full Text PDF PubMed Google Scholar), but this does not seem to be a general finding. Studies on patients with CTX have provided important information about the role of the sterol 27-hydroxylase in man. The importance of this enzyme for bile acid formation, cholesterol homeostasis, and vitamin D metabolism may, however, be different in different species. A lack of an enzyme may activate compensatory mechanisms that may be different in different species. In order to evaluate further the role of the sterol 27-hydroxylase in mammals, we have produced and characterized mice deficient of the enzyme. Oligonucleotides complementary to the putative exon 3 of the rat cyp27 were prepared (24Usui E. Noshiro M. Okuda K. FEBS Lett. 1990; 262: 135-138Crossref PubMed Scopus (112) Google Scholar, 25Su P. Rennert H. Shayiq R.M. Yamamoto R. Zheng Y.M. Addya S. Strauss III, J.F. Avadhani N.G. DNA Cell Biol. 1990; 9: 657-665Crossref PubMed Scopus (108) Google Scholar, 26Tybulewicz V.L. Crawford C.E. Jackson P.K. Bronson R.T. Mulligan R.C. Cell. 1991; 65: 1153-1163Abstract Full Text PDF PubMed Scopus (1159) Google Scholar). Oligonucleotides e (5′-AGGACAGCAGTGGTACCATCTGCG-3′) and f (5′-CTTCCAAGGCAAGGTGGTAAAGAAGA-3′) were used to amplify 197 bp containing the putative mouse exon 3 (Fig. 1). The PCR product was used to probe a mouse 129SV Genomic Library (Lambda Fix2, Stratagene, La Jolla, CA). A positive clone designated 24(1), which includes 13.1-kb mouse cyp27 gene sequences, was isolated. A targeting construct was made to replace a 71-bp Bam HI fragment in exon 8 with a neo sequence (see Ref. 27Beck E. Ludwig G. Auerswald E.A. Riess B. Schaller H. Gene (Amst.). 1982; 19: 327-336Crossref PubMed Scopus (696) Google Scholar, Fig. 1). To do so, a 7-kbBam HI fragment, which includes sequences homologous to the human CYP27 exons 2–8, was subcloned into a uniqueBam HI site of the pPNT plasmid and designated pPNT-7. A 1.1-kb Bam HI/Not I fragment, which includes the 3′ end of the putative exon 8, exon 9, and the 3′-untranslated sequences, was purified from clone 24(1), subcloned into pSC301 plasmid, and designated pSC301-S. The Bam HI/Sal I 1.1-kb band of pSC301-S was then subcloned into the pPNT-7 plasmid that was previously digested by Xho I/Not I to make pCYP27T1. In the targeting construct the deletion of a 71-bp fragment from the putative mouse cyp27 exon 8 is located upstream to the putative heme-binding site, highly conserved in human (28Cali J.J. Russell D.W. J. Biol. Chem. 1991; 266: 7774-7778Abstract Full Text PDF PubMed Google Scholar) and rabbit (29Andersson S. Davis D.L. Dahlback H. Jornvall H. Russell D.W. J. Biol. Chem. 1989; 264: 8222-8229Abstract Full Text PDF PubMed Google Scholar), and crucial for the activity of P-450 enzymes (30Gonzales F.J. Pharmacol. Rev. 1989; 40: 243-288Google Scholar). For verification, the junctions between the neo and thecyp27 sequences were sequenced and compared with the published cyp27 rat cDNA (24Usui E. Noshiro M. Okuda K. FEBS Lett. 1990; 262: 135-138Crossref PubMed Scopus (112) Google Scholar). The targeting plasmid was linearized by cleavage with Not I. ES cell cultures and electroporations were performed as described (31Piedrahita J.A. Zhang S.H. Hagaman J.R. Oliver P.M. Maeda N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4471-4475Crossref PubMed Scopus (760) Google Scholar). Ten to twelve days after electroporation, colonies resistant to 200 μg/ml G418 and to 2 μmganciclovir (Syntex, Palo Alto, CA) were picked and passaged in clonal fashion. To screen the cells with correct targeting, a probe that identifies the 1.1-kb short arm was prepared by PCR using oligonucleotide a (5′-TGGTTCCCACAAACTCCCGGATCAT-3′), which is complementary to the putative rat 5′ exon 7 sequences, and oligonucleotide T3, which is complementary to the vector short arm sequence. Southern blot analysis resulted in identification of a 9- and a 12-kb Xba I fragment for the targeted and for the non-targeted alleles, respectively. To verify the planned disruption of the gene, a probe from the putative mouse exon 3 was prepared and used in Southern blotting to identify a 5- and a 16-kb Xba I fragment from the targeted and non-targeted alleles, respectively (Fig.2 a ). Approximately 10 ES cells were injected into the blastocyte cavity of C57BL/6J embryos. Surviving blastocytes were transferred to the uteri of pseudo-pregnant CD-1 females. An average of two to three transfers were made per cell line. Chimeric animals were further bred to C57BL/6J animals to determine their germ line competency. F1 animals heterozygote for the cyp27 disruption were crossed to produce mice homozygote to the knock-outcyp27 . To genotype the F2 siblings, a double PCR reaction was performed. PCR was carried out using oligonucleotide a (5′-TGGTTCCCACAAACTCCCGGATCAT-3′) mapped to the 5′ end of the putative exon 7, oligonucleotide b (5′-CCATAGCCAAAGGGCACAGAGCCAA-3′) mapped to the 3′ end of the putative exon 8, and oligonucleotide c (5′- ATCGCATCGAGCGAGCACGTACT-3′) complementary to neo sequences (Fig. 1). The PCR products of the targeted allele and the normal gene were 1- and 0.3-kb, respectively. To avoid the out competition of the 1-kb by the 0.3-kb PCR product, a triple amount of oligonucleotide a was used. The PCR conditions were as follows: denaturation for 5 min at 94 °C followed by 35 cycles of 94 °C for 1 min, 65 °C for 1 min, and 75 °C for 1 min. To avoid PCR artifacts in genotyping, two additional, independent, reactions were carried out to identify the targeted allele and the normal allele. The oligonucleotides used for these reactions were c and d (5′-CCACCATGATATTCGGCAAGCAGG-3′, and a and b, respectively) (Fig. 3). For visualization of cyp27 mRNA the following protocol was used. RNA was extracted from mice livers using Trizol reagent (Life Technologies, Inc.), quantified by spectrophotometry and visualized following electrophoresis on a 1.5% agarose gel stained by ethidium bromide. Twenty μg of total RNA were loaded on a 1.6% agarose gel, electrophoresed, and blotted as described (32McMaster G.K. Carmichael G.G. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4835-4838Crossref PubMed Scopus (1727) Google Scholar). The blots were hybridized using a mousecyp27 exon 3 or a Syrian Hamster HMG-CoA reductase cDNA probe labeled by nick translation (Life Technologies, Inc.). For visualization and quantification of cyp7 mRNA the following protocol was used. Total cellular RNA was isolated with the UltraspecTM RNA Isolation System (Biotecx Laboratories, Houston, TX). Electrophoresis of total RNA and poly(A)+ RNA in agarose gels containing formaldehyde and blotting of the separated RNA onto nylon membranes (Hybond, Amersham Pharmacia Biotech, UK) was carried out by standard procedures (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). For the hybridization, cDNA probes for the rat cyp7 and human actin were used, labeled with 32P with the use of Pharmacia Oligolabeling kit (Amersham Pharmacia Biotech, Uppsala, Sweden). Hybridization was according to Gehring et al. (34Gehring M. Shiels B.R. Northemann W. de Bruijn M.H.L. Kan C.C. Chain A.C. Noonan D.J. Fey C.C. J. Biol. Chem. 1987; 262: 446-454Abstract Full Text PDF PubMed Google Scholar). The blots were exposed to Fuji New x-ray films at −70 °C. Semi-quantitative analysis of the relative amount of mRNA was estimated by densitometry. Livers were homogenized in a lysis buffer containing 20 mmol of Tris-HCl, pH 7.7, 1 mmol of EDTA, and 0.25m sucrose and centrifuged at 1000 × g for 5 min. The supernatant was precipitated at 5000 × g for 10 min and the mitochondrial fraction resuspended in the lysis buffer. The proteins were quantified using the Bio-Rad protein assay (Bio-Rad, Munchen, Germany). A total of 75 μg of protein was loaded on a 10% SDS-polyacrylamide gel and electrophoresed for 12 h at 15 mA. Electroblotting to cellulose nitrate (Schleicher & Schuell, Dassel, Germany) was carried out overnight at 20 V. Blocking of the membrane was carried out in a buffer containing 1.34 mNaCl, 0.03 m KCl, and 0.25 m Tris (TBS buffer) for 5 h at 4 °C. Incubation with primary polyclonal antibody directed against the mouse sterol 27-hydroxylase, extracted from rabbit serum (a gift from Dr. David W. Russell), was diluted 1/1000 in the blocking buffer (TBS) that also contained 0.05% Nonidet P-40 (Sigma). Incubation was carried out overnight at 4 °C. The filter was washed four times for 15 min each in a TBS buffer containing 0.1% SDS, 0.1% Nonidet P-40, and 0.5% deoxycholic acid sodium salt (Sigma-Aldrich Chemie, GmbH). Incubation with 1/2000 secondary antibody, anti-rabbit IgG horseradish peroxidase-linked whole antibody from donkey (Amersham Pharmacia Biotech, Buckinghamshire, UK) was carried-out for 2 h at room temperature. Development was carried out using the Amersham ECL detection reagents (Amersham Pharmacia Biotech, Buckinghamshire, UK). The film was exposed for 3 s and developed. Animals were maintained at the Hadassah-Hebrew University animal facility in a specific pathogen-free unit. Breeding pairs were set up to provide normal heterozygote and homozygote siblings of the targeted cyp27 −/−mice. Weaning was carried out at the age of 3 weeks. The animals were genotyped and grouped. Each group consisted of four to six male littermates. At the end of the experiment the animals were sacrificed, and their plasma and bile were collected and gross pathology and histology were performed. For histological processing the tissue specimens were fixed in 10% buffered formalin, dehydrated, and paraffin-embedded. Sections 2- to 3-μm thick were cut with a microtome (Leica RM2155, Germany). The sections were deparaffinized and rehydrated before hematoxylin/eosin and van Gieson's elastica stainings and periodic acid-Schiff reaction for examination in a light microscope (Olympus, Tokyo, Japan). Van Gieson's elastica was used for combined staining of elastic substrates and connective tissue. After rehydration, the sections were stained for 15 min in 0.5% resorcine/fuchsin solution in 70% ethanol. Thereafter, the sections were rinsed in distilled water, differentiated in 96% ethanol, and transferred to Weigert's iron-hematoxylin (nuclear staining) for 10 min. The sections were rinsed in distilled water, differentiated in HCl/ethanol, and rinsed in tap water for 30 min. In a third step the sections were stained in a picric acid/thiazine red mixture (10:0.2) for 10 min, rinsed in distilled water containing picric acid, and dehydrated and coverslipped by an automated coverslipper (Sakura). Periodic acid-Schiff reaction was used to demonstrate poly- and mucopolysaccharides and muco- and glycoproteins. After rehydration the sections were put into 0.5% periodic acid for 5 min at room temperature, rinsed in distilled water, and transferred to fuchsin/sulfurous acid (15 min at room temperature). The sections were washed three times in 2− water, tap water (5 min), counterstained with hemalaun, rinsed in tap water, dehydrated, and coverslipped as described above. Heart and aorta were processed separately for lipid staining with Sudan III. The heart was taken out, fixed in 10% buffered formalin for at least 48 h, incubated at 37 °C in 5% gelatin for 2.5 h, in 10% gelatin for 2.5 h and in 20% gelatin overnight. The hearts were put into a gelatin block and frozen in isopentane (−35 °C) for further processing in a cryostat (Leica, Frigocut). In brief, the hearts were cut in 10-μm thickness until the 3-valve cusps at the junction of the aorta to the heart and an aorta that was round in shape was seen. Several sections were taken at this level, stained with Sudan III, and counterstained with hemalaun. The sections were examined under a light microscope. The whole aorta was fixed in 10% buffered formalin, rinsed in tap water, cut open, blocked with needles, and stained with Sudan III. The aortas were examined for atherosclerotic plaques. A total of six animals were examined by Pathology Associates Int. (Frederick, MD, project no. 3116-101 and 97-111-09), and 18 mice were analyzed at the Max-Delbrück-Center (Berlin, Germany). Vitamin A and vitamin E levels were analyzed by high pressure liquid chromatography as described (35Catagnani G.L. Bieri J.G. Clin. Chem. 1983; 29: 708-712Crossref PubMed Scopus (526) Google Scholar). 25-Hydroxyvitamin D was analyzed by a radioimmunoassay (36Belsey R. Clark M.B. Bernat M. Glowacki J. Holick M. DeLuca H.F. Potts J.T. Am. J. Med. 1974; 57: 50-56Abstract Full Text PDF PubMed Scopus (61) Google Scholar) using a kit from Nichols Institute Diagnostics. The accuracy of this method has been ascertained by a method based on isotope dilution-mass spectrometry (37Lindbäck B. Berlin T. Björkhem I. Clin. Chem. 1987; 33: 1226-1227Crossref PubMed Scopus (18) Google Scholar). 1,25-Dihydroxyvitamin D was analyzed with a radioreceptor method (38Hollis B.W. Clin. Chem. 1986; 32: 2060-2065Crossref PubMed Scopus (275) Google Scholar) using a kit from Incstar (Incstar Corp., Stillwater, MN). Cholesterol and triglycerides in serum were analyzed with standard enzymatic photometric methods with use of commercial kits (Boehringer Mannheim, Germany). Cholesterol in feces was analyzed by isotope dilution-mass spectrometry with2H6-labeled cholesterol as internal standard (39Björkhem I. Blomstrand R. Svensson L. Clin. Chim. Acta. 1974; 54: 185-193Crossref PubMed Scopus (71) Google Scholar). Cholesterol levels in tissue extract were analyzed by the same method. The tissues were frozen in liquid nitrogen, pulverized mechanically, and extracted with chloroform/methanol (2:1, v/v). In both cases, a hydrolysis step was included, and thus the method detects both free and esterified cholesterol. Oxysterols (7α-hydroxycholesterol, 24-hydroxycholesterol, and 27-hydroxycholesterol in serum and in various organs) were analyzed by isotope dilution-mass spectrometry with use of deuterium-labeled internal standards as described previously (40Dzeletovic S. Breuer O. Lund E. Diczfalusy U. Anal. Biochem. 1995; 225: 73-80Crossref PubMed Scopus (477) Google Scholar). The procedure includes a saponification step and thus the method includes both free and esterified steroid. The interassay variation in this assay is below 5% (40Dzeletovic S. Breuer O. Lund E. Diczfalusy U. Anal. Biochem. 1995; 225: 73-80Crossref PubMed Scopus (477) Google Scholar). Hydrolyzed bile acids in bile and in feces were analyzed by combined gas chromatography under the conditions described previously (41Björkhem I. Falk O. Scand. J. Clin. Lab. Invest. 1983; 43: 163-170Crossref PubMed Google Scholar) using a Hewlett-Packard 5970 mass specific detection instrument equipped with a 0.33-μm phase HP-ultra 1 column. All samples were hydrolyzed prior to extraction, methylated with diazomethane, and trimethylsialylated prior to analysis (41Björkhem I. Falk O. Scand. J. Clin. Lab. Invest. 1983; 43: 163-170Crossref PubMed Google Scholar). The samples were analyzed by the repetitive scanning method for identification of all bile acids present in the samples (cf . Fig. 5). In addition cholic acid, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid were quantitated by isotope dilution-mass spectrometry with use of deuterium-labeled internal standards and selected ion monitoring as described previously (41Björkhem I. Falk O. Scand. J. Clin. Lab. Invest. 1983; 43: 163-170Crossref PubMed Google Scholar). The other bile acids were quantitated from the chromatogram (total ion current) obtained in the analysis of material to which no internal deuterated standards had been added (Fig. 5). The peak area of the trihydroxy bile acids was compared with the peak area of cholic acid. The amount of cholic acid had been quantitated by isotope dilution-mass spectrometry in a separate analysis as described above. The peak area of the dihydroxy bile acids was compared with the peak area of deoxycholic acid. Deoxycholic acid had been quantitated by isotope dilution-mass spectrometry in a separate experiment. In a few cases the gas chromatographic peak was found to contain contaminating interfering compounds (this was the case in the assay of the small amounts of some muricholic acid isomers in the material from thecyp27 −/− mice). In these cases a specific ion at m/z 195, specific for 6-hydroxylated bile acids, was used for assay. Unhydrolyzed bile acids in bile and urine were analyzed by electrospray mass spectrometry using a Quattro triple quadropol mass spectrometer (Fig. 4). The general conditions used for pretreatment and analysis were similar to those used for analysis of bile acids by fast atom bombardment-mass spectrometry (42Egestad B. Pettersson P. Skrede S. Sjövall J. Scand. J. Clin. Lab. Invest. 1985; 45: 443-446Crossref PubMed Scopus (39) Google Scholar) The samples (a few μl of bile and 40–300 μl of urine) were diluted with water to a 1-ml volume and applied to Sep-Pak C18 columns (Waters, Milford, MA). The columns were washed with water (5–10 ml) and eluted with methanol (2 ml). After evaporation of the solvent, the samples were dissolved in acetonitrile/distilled water (1:1, v:v). Of this solution 10 μl was introduced into the mass spectrometer via loop injection with acetonitrile/water as mobile phase. This assay was performed with isotope dilution-mass spectrometry and utilization of deuterium labeled lathosterol as internal standard as described previously (43Lund E. Sisfontes L. Reihner E. Björkhem I. Scand. J. Clin. Lab. Invest. 1989; 49: 165-171Crossref PubMed Scopus (63) Google Scholar). 7β-3H-labeled 7α-hydroxycholesterol with a specific radioactivity of 70 × 106 cpm/mg was synthesized as described previously (44Danielsson H. Einarsson K. J. Biol. Chem. 1966; 241: 1449-1454Abstract Full Text PDF PubMed Google Scholar). The steroid, about 9 × 106 cpm, was dissolved in 0.2 ml of ethanol, and this solution was added to 1 ml of 0.9% NaCl (w/v) containing 1% bovine serum albumin (w/v). The solution was injected intraperitoneally in one cyp27 +/+ and onecyp27 −/− mouse. Feces was collected during three 24-h periods after this injection. The different fecal portions were refluxed with ethanol for 24 h. The ethanol was evaporated, and the material was hydrolyzed with KOH in aqueous ethanol (41Björkhem I. Falk O. Scand. J. Clin. Lab. Invest. 1983; 43: 163-170Crossref PubMed Google Scholar). The hydrolyzed alkaline material was diluted with water and extracted three times with hexane. After acidification with diluted HCl, the water phase was extracted three times with diethyl ether. Radioactivity was measured in aliquots of the hexane phase containing neutral steroids and of the ether phase containing bile acids. Southern blot analysis on genomic DNA extracted from SV129 female mice resulted in a partial restriction map of the mousecyp27 and revealed the presence of a single copy gene (data not shown). We disrupted the cyp27 gene by inserting theneo gene and deleting 71-bp Bam HI fragment from" @default.
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W2065578656 date "1998-06-01" @default.
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W2065578656 title "Markedly Reduced Bile Acid Synthesis but Maintained Levels of Cholesterol and Vitamin D Metabolites in Mice with Disrupted Sterol 27-Hydroxylase Gene" @default.
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