Markedly Reduced Bile Acid Synthesis but Maintained Levels of Cholesterol and Vitamin D Metabolites in Mice with Disrupted Sterol 27-Hydroxylase Gene*

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. 1). 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, 7␣-hydroxylation of cholesterol is the first and rate-limiting step, and 27-hydroxylation occurs at a later stage with a 7␣-hydroxylated intermediate as substrate.
In an alternative pathway, 27-hydroxylation of cholesterol is the first step in the sequence, followed by a 7␣-hydroxylation catalyzed by a specific oxysterol 7␣-hydroxylase (1). In a recently discovered minor pathway, extrahepatic sterol 27-hydroxylase converts cholesterol into 27-hydroxycholesterol or 3␤-hydroxy-5-cholestenoic acid (2,3). The latter compounds are transported to the liver and converted into bile acids. Sterol 27-hydroxylase has also 25-hydroxylase activity toward vitamin D (4) and 1␣-hydroxylase activity toward 25hydroxyvitamin D (5). Since there is also a microsomal cytochrome P-450 that catalyzes 25-hydroxylation of vitamin D (6), 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. 7 and 8). 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 (9).
It is now well documented that patients with the rare disease cerebrotendinous xanthomatosis (CTX) 1 have a deficiency of sterol 27-hydroxylase (for a review, see Ref. 10). Recently, a number of mutations have been defined in the sterol 27-hydroxylase gene of these patients (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). As a result of the enzymatic defect, patients with CTX have a reduced synthesis of bile acids, in particular chenodeoxycholic acid (10). 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 (21,22). Due to the reduced formation of bile acids, the negative feedback of the cholesterol 7␣-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 (10). Most of this formation seems to be secondary to the accumulation of an intermediate in bile acid biosynthesis, 7␣-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 (17). 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 (1)(2)(3).
Disturbances in vitamin D metabolism have been described in a few cases of CTX (23), 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.

EXPERIMENTAL PROCEDURES
Cloning of the Mouse cyp27 and Construction of the Targeting Plasmid-Oligonucleotides complementary to the putative exon 3 of the rat cyp27 were prepared (24 -26). Oligonucleotides e (5Ј-AGGACAGCAGT-GGTACCATCTGCG-3Ј) and f (5Ј-CTTCCAAGGCAAGGTGGTAAA-GAAGA-3Ј) 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 BamHI fragment in exon 8 with a neo sequence (see Ref. 27, Fig.  1). To do so, a 7-kb BamHI fragment, which includes sequences homologous to the human CYP27 exons 2-8, was subcloned into a unique BamHI site of the pPNT plasmid and designated pPNT-7. A 1.1-kb BamHI/NotI fragment, which includes the 3Ј end of the putative exon 8, exon 9, and the 3Ј-untranslated sequences, was purified from clone 24(1), subcloned into pSC301 plasmid, and designated pSC301-S. The BamHI/SalI 1.1-kb band of pSC301-S was then subcloned into the pPNT-7 plasmid that was previously digested by XhoI/NotI 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 (28) and rabbit (29), and crucial for the activity of P-450 enzymes (30). For verification, the junctions between the neo and the cyp27 sequences were sequenced and compared with the published cyp27 rat cDNA (24).
Gene Targeting and Screening of the Homologous Recombinants-The targeting plasmid was linearized by cleavage with NotI. ES cell cultures and electroporations were performed as described (31). Ten to twelve days after electroporation, colonies resistant to 200 g/ml G418 and to 2 M ganciclovir (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 (5Ј-TGGTTCCCACAAACTCCCGGATCAT-3Ј), which is complementary to the putative rat 5Ј 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 XbaI 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 XbaI fragment from the targeted and non-targeted alleles, respectively (Fig. 2a).
Generation of Germ Line Competent Chimeras and Mouse Breeding-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-out cyp27.
PCR Genotyping of the F2 Siblings-To genotype the F2 siblings, a double PCR reaction was performed. PCR was carried out using oligonucleotide a (5Ј-TGGTTCCCACAAACTCCCGGATCAT-3Ј) mapped to the 5Ј end of the putative exon 7, oligonucleotide b (5Ј-CCATAGC-CAAAGGGCACAGAGCCAA-3Ј) mapped to the 3Ј end of the putative exon 8, and oligonucleotide c (5Ј-ATCGCATCGAGCGAGCACGTACT-3Ј) 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 (5Ј-CCACCATGATATTCG-GCAAGCAGG-3Ј, and a and b, respectively) (Fig. 3).
RNA Blot Analysis-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 g of total RNA were loaded on a 1.6% agarose gel, electrophoresed, and blotted as described (32). The blots were hybridized using a mouse cyp27 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 Ultraspec TM RNA Isolation System (Biotecx Laboratories, Houston, TX). Electrophoresis of total RNA and poly(A) ϩ 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 (33). For the hybridization, cDNA probes for the rat cyp7 and human actin were used, labeled with 32 P with the use of Pharmacia Oligolabeling kit (Amersham Pharmacia Biotech, Uppsala, Sweden). Hybridization was according to Gehring et al. (34). The blots were exposed to Fuji New x-ray films at Ϫ70°C. Semi-quantitative analysis of the relative amount of mRNA was estimated by densitometry.
Immunoblot Analysis-Livers were homogenized in a lysis buffer containing 20 mmol of Tris-HCl, pH 7.7, 1 mmol of EDTA, and 0.25 M sucrose and centrifuged at 1000 ϫ g for 5 min. The supernatant was precipitated at 5000 ϫ 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 g 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 M NaCl, 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.
Pathological Analysis-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 Ϫ/Ϫ 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-m 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 Ϫ 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 (Ϫ35°C) for further processing in a cryostat (Leica, Frigocut). In brief, the hearts were cut in 10-m 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-Delbrü ck-Center (Berlin, Germany).
Vitamin Analysis-Vitamin A and vitamin E levels were analyzed by high pressure liquid chromatography as described (35). 25-Hydroxyvitamin D was analyzed by a radioimmunoassay (36) using a kit from Nichols Institute Diagnostics. The accuracy of this method has been ascertained by a method based on isotope dilution-mass spectrometry (37). 1,25-Dihydroxyvitamin D was analyzed with a radioreceptor method (38) using a kit from Incstar (Incstar Corp., Stillwater, MN).
Lipid and Steroid Analysis-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 with 2 H 6labeled cholesterol as internal standard (39). 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 (7␣-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 (40). 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% (40).
Bile Acid Analysis-Hydrolyzed bile acids in bile and in feces were analyzed by combined gas chromatography under the conditions described previously (41) using a Hewlett-Packard 5970 mass specific detection instrument equipped with a 0.33-m phase HP-ultra 1 column. All samples were hydrolyzed prior to extraction, methylated with diazomethane, and trimethylsialylated prior to analysis (41). 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 deuteriumlabeled internal standards and selected ion monitoring as described previously (41). 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 the cyp27 Ϫ/Ϫ 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 (42) The samples (a few l of bile and 40 -300 l 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 l was introduced into the mass spectrometer via loop injection with acetonitrile/ water as mobile phase.
Assay of Lathosterol-This assay was performed with isotope dilution-mass spectrometry and utilization of deuterium labeled lathosterol as internal standard as described previously (43).
Experiments with 7␤-3 H-Labeled 7␣-Hydroxycholesterol-7␤-3 H-labeled 7␣-hydroxycholesterol with a specific radioactivity of 70 ϫ 10 6 cpm/mg was synthesized as described previously (44). The steroid, about 9 ϫ 10 6 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 ϩ/ϩ and one cyp27 Ϫ/Ϫ 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 (41). 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.

Cloning of the Mouse cyp27 and Construction of the Target-
ing Plasmid-Southern blot analysis on genomic DNA extracted from SV129 female mice resulted in a partial restriction map of the mouse cyp27 and revealed the presence of a single copy gene (data not shown). We disrupted the cyp27 gene by inserting the neo gene and deleting 71-bp BamHI fragment from exon 8 (Fig. 1b) which is adjacent to the putative hemebinding site. Electroporation of the targeting vector to ES cells resulted in colonies from which extracted DNA revealed a ϳ5-kb fragment that corresponded to the targeted allele by Southern blot analysis (Fig. 2a). These cells were used to establish cyp27 knock-out mice (Figs. 1c and 2a). Northern blot analysis of RNA extracted from wild type and mutant mice revealed a 1.9 -2.4-kb band in the normal mice and 3.9-kb band in the homozygote cyp27 Ϫ/Ϫ animals (Fig. 2b). Immunoblot analysis revealed no immunoprecipitable protein in the homozygote mice (Fig. 2c). Mutant mice were generated and genotyped by using three independent PCR reactions (Fig. 3).
Pathological Analysis-At the age of 3 weeks, the mice were weaned, and at the age of 4 weeks littermates in groups of 4 -6 animals were allocated to the different cages. A total of 18 animals were used for these experiments and were fed a normal chow diet for 3 or 6 months. In general, all animals gained weight and behaved normally. No gross abnormalities were detected. Also a detailed histological analysis of all major organs and the vascular system revealed no abnormal findings. Diffuse hepatocytic cytoplasmic pallor grade 2-3 was observed, which is a normal finding in well-nourished animals.
Formation of Bile Acids in cyp27 Ϫ/Ϫ Mice-With the use of combined gas chromatography-mass spectrometry as described under "Experimental Procedures," the concentration of bile acids in a sample of hydrolyzed bile from a cyp27 Ϫ/Ϫ mouse was found to be only about 0.9 mg/ml as compared with about 8 mg/ml in a sample from a cyp27 ϩ/ϩ mouse. As shown in Table  I, cholic acid was the dominating bile acid, and the amount of trihydroxy bile acids was almost 20-fold higher than the amount of dihydroxy bile acids in both bile samples.
The alkaline hydrolysis used in the above analysis does not cleave sulfate esters and does not give information about the type of conjugates present in the bile. In order to get qualitative information about the type of conjugates, samples of unhydrolyzed bile from a cyp27 ϩ/ϩ and a cyp27 Ϫ/Ϫ mouse were also analyzed by electrospray mass spectrometry. As shown in Fig.  4, the major peak both in the analysis of the cyp27 ϩ/ϩ and in the analysis of the cyp27 Ϫ/Ϫ appeared with a m/z at 514, corresponding to taurine conjugate of trihydroxy bile acids. In addition, there was a minor peak at m/z 498, corresponding to taurine conjugate of dihydroxy bile acids. In the analysis of the bile sample from the cyp27 Ϫ/Ϫ mouse, small peaks also appeared at m/z 531 and m/z 545. Most likely, these peaks correspond to sulfate ester of dihydroxy bile acid and monodihydroxy bile acid. Interestingly, trace amounts of compounds with m/z 611, 627, and 643 were present in the bile sample from the cyp27 Ϫ/Ϫ mouse. These peaks correspond to quasimolecular ions of glucuronides of bile alcohols containing 27-carbon atoms and four, five, and six hydroxyl groups (42). No such compounds could be detected in the analysis of the bile sample from the cyp27 ϩ/ϩ mouse.
That the production of bile acids was markedly decreased in the cyp27 Ϫ/Ϫ mice was also confirmed by combined gas chromatography-mass spectrometry of feces obtained from four cyp27 ϩ/ϩ and four cyp27 Ϫ/Ϫ mice ( Fig. 5 and Table I). As shown in Table I, the excretion of bile acids in feces was about 78 g/g in the cyp27 Ϫ/Ϫ mouse and about 430 g/g in the cyp27 ϩ/ϩ mouse. The composition of the fecal bile acids was similar in the two groups, although the relative amount of dihydroxy bile acids was somewhat higher in the cyp27 Ϫ/Ϫ group (about 50%) as compared with the cyp27 ϩ/ϩ group (about 30%). Bile acids in feces were also analyzed in a cyp27 ϩ/Ϫ mouse. The amount of bile acids (about 400 g/g feces) as well as the composition was very similar to those found in feces of cyp27 ϩ/ϩ mice.
In order to further ascertain that there is a reduced formation of bile acids in cyp27 Ϫ/Ϫ mice, one cyp27 Ϫ/Ϫ mouse and one cyp27 ϩ/ϩ mouse was injected with the same amount of 7␤-3 Hlabeled 7␣-hydroxycholesterol. Since 7␣-hydroxycholesterol is an intermediate in bile acid biosynthesis occurring after the rate-limiting step, a rapid formation of bile acids from this compound can be expected in normal mice. In accordance with this, 1.6 ϫ 10 6 cpm were found in the bile acid containing ether extract of the feces collected during 72 h after the injection of 9 ϫ 10 6 cpm in the cyp27 ϩ/ϩ mouse. The hexane extract containing neutral steroids contained 0.4 ϫ 10 6 cpm. Only 0.2 ϫ 10 6 cpm were found in the bile acid containing extract from the cyp27 Ϫ/Ϫ mouse and 1.0 ϫ 10 6 cpm in the extract containing neutral steroids. The formation of radioactive bile acids from 7␤-3 H-labeled 7␣-hydroxycholesterol in the cyp27 Ϫ/Ϫ mouse  3. PCR-based genotyping. Lanes 1-3 represent the results of a PCR reaction that included oligonucleotides a, b, and c (Fig. 1c). Oligonucleotides a and b amplify the normal allele and a and c the targeted allele. Lanes 4 -6 represent the results of PCR amplification of the targeted allele only, using oligonucleotides c and d (Fig. 1c, neo  sequences). Lanes 7-9 represent the results of PCR amplification of the wild type allele only, using oligonucleotides a and b (Fig. 1c). M, molecular weight marker; X, HaeIII-digested DNA. The results for the wild type (WT) and targeted (T) alleles are shown.
was thus only about 13% of that in the cyp27 ϩ/ϩ mouse. It should be pointed out, however, that due to the expanded pool of 7␣-hydroxycholesterol in the cyp27 Ϫ/Ϫ mice (Table III) The possibility that bile acids were excreted in urine was also tested with electrospray mass spectrometry of un-hydrolyzed urine samples from cyp27 ϩ/ϩ and cyp27 Ϫ/Ϫ mice. No significant amounts of bile acids were seen. Small peaks at m/z 627, 643, and 657 were seen in the analysis of urine from cyp27 Ϫ/Ϫ mice but not from cyp27 ϩ/ϩ mice. The latter peaks are likely to be due to glucuronides of bile alcohols containing 27 carbon atoms and five, six, and seven hydroxyl groups (42) (cf. above).
The low production of bile acids is likely to result in an up-regulation of the cholesterol 7␣-hydroxylase due to a reduced negative feedback suppression of this enzyme. In accordance with this, the concentration of 7␣-hydroxycholesterol in the liver, the kidney, and the brain was markedly higher in the cyp27 Ϫ/Ϫ mice than in the cyp27 ϩ/Ϫ and in the cyp27 ϩ/ϩ mice (Table II). It is well established that circulating levels of 7␣hydroxycholesterol reflect the activity of the cholesterol 7␣- hydroxylase in the liver (45). The concentration of circulating 7␣-hydroxycholesterol was found to be 4 -10-fold higher in the cyp27 Ϫ/Ϫ mice than in the two other genotypes (Table III).
That the cholesterol 7␣-hydroxylase was up-regulated was also confirmed by RNA blot analysis (data not shown). Thus the ratio between the cyp7 and the actin mRNA was 2.7 Ϯ 0.5 (n ϭ 7) and 0.3 Ϯ 0.1 (n ϭ 6), in the livers of the cyp27 Ϫ/Ϫ and the cyp27 ϩ/ϩ mice, respectively.
Cholesterol Synthesis-The reduced synthesis of bile acids in the knock-out mice leads to reduced absorption of cholesterol, which may lead to a compensatory increased synthesis. The up-regulation of the cholesterol 7␣-hydroxylase can also be expected to lead to a secondary up-regulation of the rate-limiting enzyme in cholesterol synthesis, HMG-CoA reductase (46 -48). In accordance with this, the hepatic levels of HMG-CoA reductase mRNA was found to be 2-3-fold higher in the knock-out mice than in the wild type (data not shown).
It is well documented that up-regulation of HMG-CoA reductase in the liver leads to increased levels of the precursor lathosterol in the circulation (49). The level of lathosterol in the circulation of three cyp27 Ϫ/Ϫ mice was found to be 769 Ϯ 56 ng/ml (mean Ϯ S.E.) as compared with 231 Ϯ 90 ng/ml in three cyp27 ϩ/ϩ mice (p Ͻ 0.01, Student's t test).
Circulating Levels of Fat-soluble Vitamins-As shown in Table IV, the levels of vitamin A were about the same in the wild type and in the cyp27 Ϫ/Ϫ mice. Vitamin E levels in the cyp27 Ϫ/Ϫ mice were somewhat lower than those of the wild type, but this difference was not significant from a statistical point of view. Levels of 25-hydroxyvitamin D were clearly higher in the cyp27 Ϫ/Ϫ mice than in the wild type (p Ͻ 0.01, Student's t test). Levels of 1,25-dihydroxyvitamin D were similar in the two groups of mice.
Circulating Levels of Cholesterol and Triglycerides, Fecal Excretion of Cholesterol-The circulating levels of cholesterol (total cholesterol) varied between 1.5 and 3.0 mmol/liter in both the wild type and the cyp27 Ϫ/Ϫ mice on a normal diet. The triglyceride levels in the three groups of mice varied between 0.2 and 0.8 mmol/liter.
The very low production of bile acids in the cyp27 Ϫ/Ϫ mice could be expected to result in increased fecal excretion of fat in feces. The excretion of cholesterol was 3.4 Ϯ 0.3 (n ϭ 5) and  1.9 Ϯ 0.2 mg/g (n ϭ 5) feces in the cyp27 Ϫ/Ϫ and the cyp27 ϩ/ϩ mice, respectively (p ϭ 0.05). Levels of Cholestanol in Liver and Circulation-The ratio between cholestanol and cholesterol was found to be 0.009 Ϯ 0.003, 0.011 Ϯ 0.003, and 0.016 Ϯ 0.006 in livers of the cyp27 Ϫ/Ϫ , the cyp27 ϩ/Ϫ , and the cyp27 ϩ/ϩ mice, respectively. Similar ratios were found in the circulation (not shown).

Consequences of the Lack of Sterol 27-Hydroxylase for Bile
Acid Synthesis-It is evident that a lack of the sterol 27hydroxylase has a more dramatic effect on production of bile acids in mice than in humans. In patients with CTX the enzyme deficiency leads to a markedly reduced production of chenodeoxycholic acid, whereas production of cholic acid is normal or almost normal (10). The latter production is most probably due to a microsomal 25-hydroxylase, active on 5␤cholestane-3␣,7␣,12␣-triol.
The product 5␤-cholestane-3␣,7␣,12␣,25-tetrol can be converted into cholic acid in a pathway involving cleavage of acetone from the steroid side chain (cf. above). The microsomal 25-hydroxylase is considerably more active in human liver microsomes than in rat liver microsomes (51). In preliminary experiments the activity of this enzyme was found to be about equal in rat and mouse liver. The relatively low activity of the microsomal 25-hydroxylase in mouse liver may thus explain why cyp27 Ϫ/Ϫ mice are unable to compensate for the lack of the sterol 27-hydroxylase by formation of bile acids through the alternative 25-hydroxylase mechanism.
The small amounts of bile acids found in the cyp27 Ϫ/Ϫ mice might in part have been formed by the sterol 25-hydroxylase pathway. Small amounts of bile alcohols were found in bile and urine of cyp27 Ϫ/Ϫ mice, and trace amounts of 5␤-cholestane-3␣,7␣,12␣,25-tetrol could in fact be identified in feces of one of the cyp27 Ϫ/Ϫ mice.
In contrast to humans, rat liver contains a microsomal 26hydroxylase that is able to catalyze 26-hydroxylation of 5␤cholestane-3␣,7␣,12␣-triol (52). The product, 5␤-cholestane-3␣,7␣,12␣,26-tetrol, being an isomer to 5␤-cholestane-3␣,7␣,12␣,27-tetrol, might well be further converted into cholic acid. Whether or not mouse liver microsomes contain such a 26-hydroxylase is not known with certainty. If such enzyme activity exists, it may also explain part of the small amounts of bile acids formed in the cyp27 Ϫ/Ϫ mice. At the present state of knowledge, we also cannot exclude that a sterol 24-hydroxylase present in the brain (53) may be of some importance for the conversion of cholesterol into bile acids in the cyp27 Ϫ/Ϫ mice.
In accordance with the findings in patients with CTX, the lack of the sterol 27-hydroxylase resulted in a compensatory up-regulation of the cholesterol 7␣-hydroxylase in the cyp27 Ϫ/Ϫ mice. The circulating levels of 7␣-hydroxycholesterol were thus markedly elevated, and RNA blot analysis showed about 9-fold increased concentrations of cholesterol 7␣-hydroxylase mRNA in the liver.
Formation of Cholestanol in cyp27 Ϫ/Ϫ Mice-One of the characteristics of CTX is an increased production of cholestanol (10). This production is most probably linked to the accumulation of 7␣-hydroxylated intermediates in bile acid biosynthesis, in particular 7␣-hydroxy-4-cholesten-3-one. Conversion of 7␣hydroxy-4-cholesten-3-one into cholestanol requires a critical dehydration of this steroid by a microsomal dehydratase (54). We have shown that the activity of this enzyme is severalfold higher in human liver than in rat liver (54). In preliminary experiments we showed that the activity of this dehydratase is about similar in rat and in mouse liver. The relatively low activity of this enzyme may thus explain why the cyp27 Ϫ/Ϫ mice did not have higher levels of cholestanol than the cyp27 ϩ/ϩ mice.
Consequences of the Lack of Sterol 27-Hydroxylase for Transport of Cholesterol from Peripheral Tissues-Patients with CTX are known to develop premature atherosclerosis despite normal circulating levels of cholesterol (10). It has been suggested that this could be a consequence of the lack of the sterol 27hydroxylase in macrophages in these patients (1)(2)(3). The sterol 27-hydroxylase is thus able to eliminate cholesterol from macrophages by converting it into 27-hydroxycholesterol and 3␤-hydroxy-5-cholestenoic acid that are easily excreted from the cells. The general importance of this mechanism is reflected in the very high levels of sterol 27-hydroxylase in human macrophages (1,2) and in the high flux of 27-oxygenated C 27 steroids from extrahepatic tissues to the liver in humans (3). The latter flux is likely to be of similar magnitude in mice and in humans, as judged from the relatively high levels of circulating 27-hydroxycholesterol (Table III). Under the conditions employed, however, the cyp27 Ϫ/Ϫ mice did not produce visible xanthomas or atheromas. At the present state of knowledge, it cannot be excluded that the accumulation of cholestanol and/or C 27 bile alcohols that are seen in CTX patients but not in the cyp27 Ϫ/Ϫ mice is the most critical factor for the development of xanthomas and atheromas in CTX patients.
Consequences of the Lack of the Sterol 27-Hydroxylase for Cholesterol Synthesis and Homeostasis-27-Hydroxycholesterol is known to be a potent down-regulator of HMG-CoA reductase in cultured cells (7,8). The importance of this mechanism is, however, controversial. If the sterol 27-hydroxylase has a key role in cholesterol homeostasis, a lack of the enzyme would be expected to be associated with a markedly increased synthesis of cholesterol and elevated circulating levels of cholesterol in the circulation. CTX patients are, however, known to have normal circulating levels of cholesterol, and this was found to be the case also in the present cyp27 Ϫ/Ϫ mice. Patients with CTX are known to have an overall increased cholesterol synthesis, most probably secondary to the up-regulated cholesterol 7␣-hydroxylase (10). That the increased cholesterol synthesis in the present sterol 27-hydroxylase-deficient mice is secondary to the up-regulated cholesterol 7␣-hydroxylase seems likely in view of the finding that the synthesis of the latter enzyme was increased much more than the synthesis of HMG-CoA reductase. The up-regulated hepatic HMG-CoA reductase may be a compensation for a consumption of cholesterol due to the highly up-regulated cholesterol 7␣-hydroxylase (cf. Ref. 48).
Consequences of the Lack of the Sterol 27-Hydroxylase for Metabolism of Vitamin D-Sterol 27-hydroxylase is able to 25-hydroxylate vitamin D (4). Since the liver mitochondrial fraction of a patient with CTX has been found to possess some 25-hydroxylase activity toward vitamin D (55) and since the microsomal fraction of liver homogenate also contains this activity (6), the relative importance of the sterol 27-hydroxylase for this reaction is difficult to evaluate. Reduced bone density and reduced circulating levels of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D have also been reported in some  (23). Other patients have normal levels of 25hydroxyvitamin D, however (10). 2 A serum pool from three cyp27 Ϫ/Ϫ mice had somewhat higher concentrations of 25-hydroxyvitamin D than a serum pool from three cyp27 ϩ/ϩ mice. In view of this, sterol 27-hydroxylase seems to be of little importance for 25-hydroxylation of vitamin D in mice. Recently it was shown that sterol 27-hydroxylase has some 1␣-hydroxylase activity toward 25-hydroxyvitamin D 3 (5). The circulating concentration of 25-dihydroxyvitamin D was, however, somewhat higher in cyp27 Ϫ/Ϫ mice than in cyp27 ϩ/ϩ mice, whereas the corresponding concentrations of 1,25-dihydroxyvitamin D were similar in the two groups of mice. Thus it seems less likely that sterol 27-hydroxylase is of importance in formation of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D in this species.
Consequences of the Reduced Formation of Bile Acids in cyp27 Ϫ/Ϫ Mice-Surprisingly, the markedly reduced production of bile acids in the cyp27 Ϫ/Ϫ mice was not associated with any symptoms of malabsorption. The content of cholesterol and fatty acids in feces was thus only moderately increased, and the circulating levels of the fat-soluble vitamins A and E were normal or only slightly reduced. It is interesting to compare this situation with the situation in mice with a disruption of the cyp7 (56). During the first 4 weeks of life, these mice had a severe fat malabsorption with very low circulating levels of 25-hydroxyvitamin D 3 and vitamin E and high content of lipids in stool. After this period a normalization occurred, most probably due to induction of an oxysterol 7␣-hydroxylase in the liver. The adult cyp27 Ϫ/Ϫ mice had an excretion of bile acids in stool that was reduced by about 80% as compared with the wild type. Evidently, this low degree of production of bile acids is sufficient to prevent fat malabsorption.
Is the Cyp 27 Ϫ/Ϫ Mouse Suitable as an Animal Model for CTX?-It is evident that a disruption of the cyp27 Ϫ/Ϫ gene in a mouse on a normal diet does not lead to the metabolic, neurologic, and vascular disturbances found in patients with a corresponding genetic defect. The compensatory mechanism in humans (activation of the 25-hydroxylase pathway) and the consequences of accumulation of bile acid intermediates (accumulation of cholestanol) were not observed in the cyp27 Ϫ/Ϫ mice. It is possible that the enzymatic defect in the cyp27 Ϫ/Ϫ mice may be more important when increasing the dietary intake of cholesterol. In preliminary experiments an increased death rate was observed for cyp27 Ϫ/Ϫ mice when fed an atherogenic diet. No explanation for this finding has been obtained thus far, however. Experiments with the goal of clarifying the importance of the sterol 27-hydroxylase in mice with additional genetic defects and under different dietary conditions are now in progress.