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• W2007172615 abstract "Past experiments and current paradigms of cholesterol homeostasis suggest that cholesterol 7α-hydroxylase plays a crucial role in sterol metabolism by controlling the conversion of cholesterol into bile acids. Consistent with this conclusion, we show in the accompanying paper that mice deficient in cholesterol 7α-hydroxylase (Cyp7−/− mice) exhibit a complex phenotype consisting of abnormal lipid excretion, skin pathologies, and behavioral irregularities (Ishibashi, S., Schwarz, M., Frykman, P. K., Herz, J., and Russell, D. W. (1996) J. Biol. Chem. 261, 18017-18023). Aspects of lipid metabolism in the Cyp7−/− mice are characterized here to deduce the physiological basis of this phenotype. Serum lipid, cholesterol, and lipoprotein contents are indistinguishable between wild-type and Cyp7−/− mice. Vitamin D3 and E levels are low to undetectable in knockout animals. Stool fat content is significantly elevated in newborn Cyp7−/− mice and gradually declines to wild-type levels at 28 days of age. Several species of 7α-hydroxylated bile acids are detected in the bile and stool of adult Cyp7−/− animals. A hepatic oxysterol 7α-hydroxylase enzyme activity that may account for the 7α-hydroxylated bile acids is induced between days 21 and 30 in both wild-type and deficient mice. An anomalous oily coat in the Cyp7−/− animals is due to the presence of excess monoglyceride esters in the fur. These data show that 7α-hydroxylase and the pathway of bile acid synthesis initiated by this enzyme are essential for proper absorption of dietary lipids and fat-soluble vitamins in newborn mice, but not for the maintenance of serum cholesterol and lipid levels. In older animals, an alternate pathway of bile acid synthesis involving an inducible oxysterol 7α-hydroxylase plays a crucial role in lipid and bile acid metabolism. Past experiments and current paradigms of cholesterol homeostasis suggest that cholesterol 7α-hydroxylase plays a crucial role in sterol metabolism by controlling the conversion of cholesterol into bile acids. Consistent with this conclusion, we show in the accompanying paper that mice deficient in cholesterol 7α-hydroxylase (Cyp7−/− mice) exhibit a complex phenotype consisting of abnormal lipid excretion, skin pathologies, and behavioral irregularities (Ishibashi, S., Schwarz, M., Frykman, P. K., Herz, J., and Russell, D. W. (1996) J. Biol. Chem. 261, 18017-18023). Aspects of lipid metabolism in the Cyp7−/− mice are characterized here to deduce the physiological basis of this phenotype. Serum lipid, cholesterol, and lipoprotein contents are indistinguishable between wild-type and Cyp7−/− mice. Vitamin D3 and E levels are low to undetectable in knockout animals. Stool fat content is significantly elevated in newborn Cyp7−/− mice and gradually declines to wild-type levels at 28 days of age. Several species of 7α-hydroxylated bile acids are detected in the bile and stool of adult Cyp7−/− animals. A hepatic oxysterol 7α-hydroxylase enzyme activity that may account for the 7α-hydroxylated bile acids is induced between days 21 and 30 in both wild-type and deficient mice. An anomalous oily coat in the Cyp7−/− animals is due to the presence of excess monoglyceride esters in the fur. These data show that 7α-hydroxylase and the pathway of bile acid synthesis initiated by this enzyme are essential for proper absorption of dietary lipids and fat-soluble vitamins in newborn mice, but not for the maintenance of serum cholesterol and lipid levels. In older animals, an alternate pathway of bile acid synthesis involving an inducible oxysterol 7α-hydroxylase plays a crucial role in lipid and bile acid metabolism. Two pathways of bile acid biosynthesis have been described in the mammalian liver. One pathway is initiated in the endoplasmic reticulum of the hepatocyte by the enzyme cholesterol 7α-hydroxylase (referred to hereafter as 7α-hydroxylase; cholesterol 7α-monooxygenase (EC)), which converts cholesterol (cholest-5-en-3β-ol) into 7α-hydroxycholesterol (cholest-5-ene-3β,7α-diol). Subsequent enzymatic steps lead to the formation of the primary bile acids cholic acid and chenodeoxycholic acid (reviewed in 1Russell D.W. Setchell K.D.R. Biochemistry. 1992; 31: 4737-4749Google Scholar). A second pathway is initiated in the mitochondria by the enzyme sterol 27-hydroxylase, which converts cholesterol into 27-hydroxycholesterol (cholest-5-ene-3β,27-diol) (2Axelson M. Sjövall J. J. Steroid Biochem. 1990; 36: 631-640Google Scholar). This intermediate is acted on by an oxysterol 7α-hydroxylase to form 7α,27-dihydroxycholesterol (cholest-5-ene-3β,7α,27-triol) (3Björkhem I. J. Lipid Res. 1992; 33: 455-471Google Scholar, 4Toll A. Wikvall K. Sudjana-Sugiaman E. Kondo K. Björkhem I. Eur. J. Biochem. 1994; 224: 309-316Google Scholar, 5Axelson M. Shoda J. Sjövall J. Toll A. Wikvall K. J. Biol. Chem. 1992; 267: 1701-1704Google Scholar, 6Payne D.W. Shackleton C. Toms H. Ben-Shlomo I. Kol S. deMoura M. Strauss J.F. Adashi E.Y. J. Biol. Chem. 1995; 270: 18888-18896Google Scholar, 7Zhang J. Larsson O. Sjövall J. Biochim. Biophys. Acta. 1995; 1256: 353-359Google Scholar), which is subsequently converted into primary bile acids. The pathway initiated by 7α-hydroxylase is thought to be the major route by which bile acids are synthesized in the liver. This assumption arises in part because the 7α-hydroxylase pathway was discovered first (8Danielsson H. Einarsson K. Johansson G. Eur. J. Biochem. 1967; 2: 44-49Google Scholar) and because the level of 7α-hydroxylase enzyme activity is tightly controlled by feedback regulation (1Russell D.W. Setchell K.D.R. Biochemistry. 1992; 31: 4737-4749Google Scholar). The importance of this pathway is underscored by the finding that expression of an exogenous 7α-hydroxylase gene in hamsters via infection with a recombinant adenovirus leads to a marked increase in bile acid formation (9Spady D.K. Cuthbert J.A. Willard M.N. Meidell R.S. J. Clin. Invest. 1995; 96: 700-709Google Scholar). To gain further insight into the role of 7α-hydroxylase in bile acid metabolism, a line of mice deficient in this enzyme was created by gene targeting methods (10Ishibashi S. Schwarz M. Frykman P.K. Herz J. Russell D.W. J. Biol. Chem. 1996; 261: 18017-18023Google Scholar). Young 7α-hydroxylase-deficient mice (Cyp7−/−) exhibit a complex phenotype consisting of an increased rate of postnatal death, fat malabsorption, wasting, skin abnormalities, and vision problems (10Ishibashi S. Schwarz M. Frykman P.K. Herz J. Russell D.W. J. Biol. Chem. 1996; 261: 18017-18023Google Scholar). The absence of bile acids in newborn animals combined with fat-soluble vitamin deficiency is considered the most likely explanation for this phenotype. A peculiar feature of murine 7α-hydroxylase deficiency is that the phenotype is only present in newborn mice: once a deficient animal reaches the age of ∼3 weeks, symptoms wane to the point that adult animals are indistinguishable from wild-type mice (10Ishibashi S. Schwarz M. Frykman P.K. Herz J. Russell D.W. J. Biol. Chem. 1996; 261: 18017-18023Google Scholar). We now explain this phenotype by showing that the mitochondrial bile acid pathway is not present at birth, but appears at day 21 in both wild-type and Cyp7−/− mice, thereby obviating the requirement for 7α-hydroxylase. Induction correlates with the appearance of oxysterol 7α-hydroxylase activity in the liver and results in the formation of bile with an altered composition of bile acids relative to animals in which both pathways are functioning. By studying the levels of vitamins D3 and E, we have formulated the hypothesis that the bile acid products of the mitochondrial pathway are not as efficient as those of the endoplasmic reticulum pathway in mediating vitamin E absorption. Thus, the requirement for two pathways may reflect a need to synthesize bile acids of diverse chemical structures in order to ensure maximum solubilization of different dietary fats and vitamins. Bile was drawn from the gallbladders of mice euthanized with sodium pentobarbital. Samples were stored frozen at −20°C until analyzed. Blood for lipoprotein, cholesterol, and triglyceride analysis was drawn by cardiac puncture or exsanguination via the ascending carotid artery and clotted at room temperature for 30 min. The sample was centrifuged at 16,000 × g for 50 min, and the serum was decanted from the pelleted blood cells. The levels of cholesterol, triglyceride, and lipoprotein were determined as described previously (11Ishibashi S. Brown M.S. Goldstein J.L. Gerard R.D. Hammer R.E. Herz J. J. Clin. Invest. 1993; 92: 883-893Google Scholar). Vitamin E levels were measured in epididymal or ovarian fat samples using high pressure liquid chromatography methods as described previously (12Traber M.G. Kayden H.J. Am. J. Clin. Nutr. 1987; 46: 488-495Google Scholar). Vitamin D metabolites were assayed as described previously (13Popoff S.N. Osier L.K. Zerwekh J.E. Marks S.C. Bone (Elmsford). 1994; 15: 515-522Google Scholar, 14Sakhaee K. Baker S. Zerwekh J.E. Poindexter J. Garcia-Hernandez P.A. Pak C.Y.C. J. Urol. (Paris). 1994; 152: 324-327Google Scholar), except that bovine mammary gland vitamin D receptor was used in place of calf thymus gland vitamin D receptor. In brief, serum samples were supplemented with trace quantities of radioactive 25-hydroxyvitamin D3 to quantify the yield of this metabolite during the ensuing purification. Following extraction of vitamin D metabolites from serum with acetonitrile and back-extraction with phosphate buffer, the vitamin D metabolites were purified and separated via chromatography on C18 and silica Sep-Pak cartridges (Waters). After removing an aliquot of the purified fractions for determination of yield, assay of 25-hydroxyvitamin D3 was performed via competitive protein binding using a 1:5000 dilution of human serum (vitamin D-binding protein) as the binding agent. The sensitivity of this assay is 0.2 ng or a serum value of 1 ng/ml assuming 80% yield and a single determination. The intra- and interassay coefficients of variation are 5 and 8%, respectively. Because this purification scheme does not separate the D2 (ergocalciferol) and D3 (cholecalciferol) forms of the metabolites, the concentration measured in serum represents total vitamin D. The extraction, separation, derivatization, and analyses of bile acids in bile using gas chromatography-mass spectrometry and liquid secondary ionization mass spectrometry were carried out as described previously (15Setchell K.D.R. Yamashita H. Rodrigues C.M.P. O'Connell N.C. Kren B.T. Steer C.J. Biochemistry. 1995; 34: 4169-4178Google Scholar). Oxysterol 7α-hydroxylase activity was determined in 0.5-ml incubation mixtures containing 0.06 nmol of 25-[26,27-3H2]hydroxycholesterol (77 Ci/mmol), 250 µg of microsomal protein, 0.75 µmol of NADPH, 50 mM Tris acetate, pH 7.4, 1 mM EDTA, 2 mM dithiothreitol, and 0.03% (v/v) Triton X-100. After incubation for 15 min at 37°C, the reactions were terminated by the addition of 6 ml of methylene chloride. The organic phase was evaporated to dryness under nitrogen; the lipid pellet was dissolved in 40 µl of acetone and analyzed by thin-layer chromatography on Silica Gel LK5D 150-Å plates (Whatman) in a solvent system containing toluene/ethyl acetate (2:3). 1The compositions of all solvent systems are indicated as volumetric ratios. A sample of authentic cholest-5-ene-3β,7α,25-triol was synthesized using a modification of a method described previously (16Schenck G.O. Golinick K. Neumüller O.A. Ann. Chem. (Justus Liebigs). 1957; 603: 46-59Google Scholar, 17Schenck G.O. Neumüller O.A. Eisfeld W. Angew. Chem. Int. Ed. Engl. 1958; 19: 595Google Scholar). Briefly, cholest-5-ene-3β,25-diol was photochemically converted into 5α-hydroperoxycholest-6-ene-3β,25-diol in the presence of oxygen and hematoporphyrin. The product was purified by silica gel chromatography with a 46% yield and incubated in chloroform to allow rearrangement to the 7α-hydroperoxide derivative. This material was reduced with sodium borohydride to yield the desired product, which was purified by preparative thin-layer chromatography in a toluene/ethyl acetate (2:3) solvent system. The final yield of cholest-5-ene-3β,7α,25-triol was 3-5%. Scissors were used to barber fur from the abdomens or backs of mice. A 10-mg aliquot of fur was extracted with 1.5 ml of chloroform/methanol (2:1) for 2 h at 4°C with continuous agitation. The resulting solvent was transferred to a fresh polypropylene tube and evaporated to dryness under a nitrogen stream. The pellet was dissolved in 50 µl of chloroform/methanol (2:1) and analyzed by thin-layer chromatography on Silica Gel LK5D 150-Å plates in a solvent system containing toluene/ethyl acetate (7:3). For stool lipid analysis, 100-mg aliquots of droppings collected from animals of the indicated ages were mixed with a small amount of [carboxyl-14C]triolein (112 mCi/mmol) and dried for 1 h in a vacuum oven at 70°C. The solid matter was extracted with 2 ml of chloroform/methanol (2:1) for 30 min at 60°C, passed through a Whatman No. 1 filter, and brought to a final volume of 4 ml with chloroform/methanol (2:1). The material was back-extracted with 1 ml of H2O, and the organic phase was evaporated to dryness. The pellet was resuspended in 2 ml of chloroform/methanol (2:1) and transferred to preweighed vials. The solvent was evaporated, and the vial was taken to a constant weight by drying in a vacuum oven at 70°C. The difference in weight between the starting empty vial and the vial containing the dried lipid was the fecal lipid amount, which was expressed as a percentage of the weight of the starting fecal sample. The percent recovery of radiolabeled triolein (85-92%) was determined by subjecting the vial to scintillation counting. Gas chromatography-mass spectrometry of mouse fur lipids was performed before and after saponification as described previously (18Dzeletovic S. Breuer O. Lund E. Diczfalusy U. Anal. Biochem. 1995; 225: 73-80Google Scholar). Native lipids were converted into trimethylsilyl ethers prior to analysis (19Lund E. Boberg K.M. Byström S. Ölund J. Carlström K. Björkhem I. J. Biol. Chem. 1991; 266: 4929-4937Google Scholar). Briefly, samples were treated with pyridine/hexamethyldisilazane/chlorotrimethylsilane (3:2:1) at 60°C for 30 min. The solvent was evaporated under a stream of nitrogen, and the residue was dissolved in hexane for gas chromatography-mass spectrophotometry analyses. Saponified samples were converted into methyl esters by treatment with diazomethane (19Lund E. Boberg K.M. Byström S. Ölund J. Carlström K. Björkhem I. J. Biol. Chem. 1991; 266: 4929-4937Google Scholar), further derivatized with trimethylsilane as described above, and then subjected to chemical analysis. The normal diet was mouse/rat diet 7001 (Harlan Teklad, Madison, WI) and contained ≥4% (w/w) fat, ≥24% (w/w) protein, and ≤5% (w/w) fiber. Where indicated, this diet was supplemented with 1% (w/w) cholic acid (Sigma). Vitamin supplements (CritterVites, Mardel Labs, Glendale Heights, IL) containing both water-soluble vitamins (thiamine, 180 mg/kg; riboflavin, 300 mg/kg; pantothenic acid, 600 mg/kg; niacin, 1500 mg/kg; vitamin B12, 1500 µg/kg; vitamin B6, 150 mg/kg; folic acid, 100 mg/kg; and ascorbic acid, 9000 mg/kg) and fat-soluble vitamins (vitamin A, 300,000 IU/kg; vitamin D3, 50,000 IU/kg; vitamin E, 750 IU/kg; and menadione, 250 mg/kg) were added to water bottles at the concentration (1 g/liter) recommended by the manufacturer. Vitamin supplements were replaced on a daily basis. To determine the effects of 7α-hydroxylase deficiency on serum lipid levels, blood was sampled from animals of different genotypes and ages, and the serum lipids were analyzed. The data of Table I show that although there is wide interanimal variation, serum triglyceride and cholesterol contents are similar in wild-type and deficient mice, regardless of the age of the animals. In agreement with these measurements, the profiles of lipoprotein particles in the serum were similar in individual animals of different Cyp7 genotypes (Fig. 1). In experiments not shown, the concentrations of cholesterol in several tissues (liver, spleen, kidney, lung, and heart) were not significantly different between 15-day-old wild-type and mutant mice.Table I.Serum cholesterol and triglyceride levels in wild-type and Cyp7−/− miceAgeCyp7 genotypeCholesterolaValues represent means±S.D. derived from measurements made in the sera of 4-10 animals of the indicated age and genotype.TriglycerideaValues represent means±S.D. derived from measurements made in the sera of 4-10 animals of the indicated age and genotype.daysmg/dlmg/dl6+/+102.2 ±16.5177.6 ±104.7−/−85.6 ±22.3140.0 ±42.215+/+170.9 ±30.793.0 ±42.8−/−157.5 ±44.3151.7 ±63.323+/+88.4 ±10.054.2 ±10.7−/−99.7 ±19.682.3 ±28.060-90+/+91.5 ±7.964.4 ±7.9−/−91.7 ±5.639.7 ±7.7150-180+/+107.1 ±15.776.3 ±19.1−/−122.5 ±20.082.5 ±27.2a Values represent means±S.D. derived from measurements made in the sera of 4-10 animals of the indicated age and genotype. Open table in a new tab Many of the phenotypic characteristics of 7α-hydroxylase-deficient mice, including early death, skin abnormalities, and eye and vision problems, are reminiscent of fat-soluble vitamin deficiencies. This hypothesis is supported by the finding that these symptoms can be alleviated by supplementing the water supply of nursing mothers with a vitamin mixture (10Ishibashi S. Schwarz M. Frykman P.K. Herz J. Russell D.W. J. Biol. Chem. 1996; 261: 18017-18023Google Scholar). To determine if a fat-soluble vitamin deficiency could be directly demonstrated, the levels of vitamins D3 and E were measured in the serum and fat, respectively, of mutant and wild-type mice. The data of Table II show that Cyp7−/− mice contain low levels of vitamins D3 and E. These deficiencies are detected in mice of different ages and in nursing mothers. Vitamin supplementation alone partially restored levels of vitamins D3 and E, whereas dietary supplementation with vitamins and a bile acid (cholic acid) more fully restored vitamin levels (Table II).Table II.Serum vitamin D3 and tissue vitamin E levels in wild-type and Cyp7−/− miceAgeaSerum or fat tissue was pooled from 3-12 male and female mice to derive the values shown for the 6-,16-,and 23-day time points.,bValues for 34-38 and 120-180-day mice represent means±S.E. derived from three to five animals.Cyp7 genotypeSerum vitamin D3c25-Hydroxyvitamin D3 levels were measured in serum as described under “Experimental Procedures.”Tissue vitamin EdVitamin E levels were measured in epididymal or ovarian fat pads as described under “Experimental Procedures.”ChowChow+vitaminsChow+vitamins, cholic acidChowChow+vitaminsChow+vitamins, cholic aciddaysng/mlng/mg triglyceride6+/+20.0—e—, not done.————−/−2.05.012.0———16+/+18.0——11.6——−/−3.74.09.00.3<0.112.023+/+8.0——24.4——−/−1.04.09.0<0.1<0.153.334-38+/+17.7 ±1.1——10.4 ±2.0——−/−15.7 ±2.4——5.6 ±3.6——120-180+/+17.5 ±2.5——90.7 ±25.4——−/−20.0 ±3.311.0±4.428.5±2.54.4 ±4.411.6±2.5146.1±91.8a Serum or fat tissue was pooled from 3-12 male and female mice to derive the values shown for the 6-,16-,and 23-day time points.b Values for 34-38 and 120-180-day mice represent means±S.E. derived from three to five animals.c 25-Hydroxyvitamin D3 levels were measured in serum as described under “Experimental Procedures.”d Vitamin E levels were measured in epididymal or ovarian fat pads as described under “Experimental Procedures.”e —, not done. Open table in a new tab A second characteristic feature of newborn 7α-hydroxylase-deficient mice is their excretion of clay-colored stools, which are reminiscent of fat malabsorption (steatorrhea). To confirm this diagnosis, the stool fat content was monitored as a function of age in Cyp7−/− mice. The data of Fig. 2 show that newborn mice have enormously elevated levels of fat in their stools and that this elevation persists through approximately postnatal day 22, at which time the stool fat content begins to decrease and eventually (by day 28) approximates that of wild-type animals. Weaning took place on day 30 in these experiments; thus, the decline in fat content occurred while the animals were maintained on a high fat diet (mother's milk). The reduction in stool fat content on or about postnatal day 22 coincides with the time at which the survival rate of mutant mice is dramatically increased (10Ishibashi S. Schwarz M. Frykman P.K. Herz J. Russell D.W. J. Biol. Chem. 1996; 261: 18017-18023Google Scholar). Those few animals that reach this age thereafter experience a normal life span. These findings are suggestive of a major change in bile acid metabolism occurring around postnatal day 22 in the Cyp7−/− mice. The chemical composition of bile from adult wild-type and Cyp7−/− animals (∼3 months of age) was examined next. Animals were maintained on normal unsupplemented chow for ≥4 weeks; the major bile duct was cannulated; and bile was collected over a period of 30-60 min from two mice of each genotype. The withdrawn samples were analyzed by gas chromatography-mass spectrometry to identify individual bile acids. The results of these analyses are shown in Table III. The concentration of bile acids in the bile of wild-type and mutant mice was similar and ranged from 9.9 to 14.6 mM. Bile from wild-type animals contained at least 11 separable and measurable bile acid derivatives, of which cholic acid and β-muricholic acid were the predominant species, accounting together for ∼75% of the total bile acids. In the Cyp7−/− mice, the concentration of cholic acid, but not β-muricholic acid, was decreased by more than half relative to that found in the wild-type animals (Table III). In addition, several bile acids were increased in concentration relative to levels in wild-type animals. For example, hyodeoxycholic acid (3α,6α-dihydroxy-5β-cholanoic acid) was not detected in wild-type bile, but this compound represented 14-25% of the total bile acid in the Cyp7−/− animals. Similarly, chenodeoxycholic acid and α-muricholic acid were detected only in the mutant mice (Table III).Table III.Analysis of bile acids in bile of individual wild-type and Cyp7−/− adult miceRetention timeaBile acids are listed based on retention times as methyl ester-trimethylsilyl ethers relative to a homologous series of n-alkanes, referred to as the methylene unit (MU) value.Bile acidbChemical structures were established by electron ionization-gas chromatography-mass spectrometry.Cyp7 genotype+/++/+−/−−/−MUµmol/liter31.813,12-Dihydroxy bile acid31242510718932.003α,7α,12α-Trihydroxy-5α-cholanoic acid (allo-cholic acid)48265117423632.133α,7α-Dihydroxy-5β-cholanoic acid (chenodeoxycholic acid)NDcND, not detected.ND43135032.183α,6β,7α-Trihydroxy-5β-cholanoic acid (α-muricholic acid)NDND76667232.233α,7α,12α-Trihydroxy-5β-cholanoic acid (cholic acid)671076091918243432.293α,6α-Dihydroxy-5β-cholanoic acid (hyodeoxycholic acid)NDND2486171332.513α,7β-Dihydroxy-5β-cholanoic acid (ursodeoxycholic acid)15030416816232.843α,7α,12α-Trihydroxy-5β-homocholanoic acid (homocholic acid)155178NDND33.0712-Oxo-3α-hydroxy-5β-cholanoic acid15721789974633.213α,6β,7β-Trihydroxy-5β-cholanoic acid (β-muricholic acid)177831311717306033.523α,7β,12α-Trihydroxy-5α-cholanoic acid13223717114633.72Oxodihydroxy bile acid27638016313933.807-Oxo-3α,12α-Dihydroxy-5β-cholanoic acid260290ND34034.293α,6α,7β-Trihydroxy-5β-cholanoic acid (ω-muricholic acid)55512039021714Total bile acids10,96714,625990211,901a Bile acids are listed based on retention times as methyl ester-trimethylsilyl ethers relative to a homologous series of n-alkanes, referred to as the methylene unit (MU) value.b Chemical structures were established by electron ionization-gas chromatography-mass spectrometry.c ND, not detected. Open table in a new tab The data of Table IV summarize the results obtained after chemical analyses of bile acids in stool samples of adult normal and Cyp7−/− animals (∼4 months of age). Feces were collected for a period of several days from five individual animals of each genotype, weighed, and then extracted and analyzed for bile acids. The average weight of stool collected from the wild-type mice was 1.32 ± 0.12 g/day (mean ± S.E.) versus 1.35 ± 0.05 g/day for the Cyp7−/− mice. The concentration of total bile acids in the droppings of wild-type mice was 1095.8 ± 88.9 µg/g of feces, whereas the droppings from Cyp7−/− mice contained 369.8 ± 18.2 µg/g of feces (Table IV). The diminished bile acid concentration in the stool samples of the mutant animals reflected a uniform decline in almost all of the individual bile acid species detected in the analyses (Table IV). For example, deoxycholic acid, which is the most abundant bile acid in mouse stool, was reduced from 316.2 ± 28.6 µg/g in the wild-type animals to 47.4 ± 12.6 µg/g in the Cyp7−/− animals (Table IV).Table IV.Analysis of fecal bile acids in individual wild-type and Cyp7−/− adult miceRetention timeaBile acids are listed based on their retention times as methyl ester-trimethylsilyl ethers relative to a homologous series of n-alkanes, referred to as the methylene unit (MU) value.Bile acidbChemical structures were determined by electron ionization-gas chromatography-mass spectrometry.Cyp7 genotype+/++/++/++/++/+−/−−/−−/−−/−−/−MUµg/g feces31.183α-Hydroxy-5β-cholanoic acid (lithocholic acid)1223262231866ND431.61Unknown bile acidNDcND, not detected.12231818NDND4NDND31.763,12-Dihydroxy bile acid2127263432365ND431.883α-12α-Dihydroxy-5β-cholanoic acid (deoxycholic acid)242276319411333417378103532.003α,7α,12α-Trihydroxy-5α-cholanoic acid (allo-cholic acid)181111ND9NDND68832.153α,7α-Dihydroxy-5β-cholanoic acid (chenodeoxycholic acid)ND811ND108667432.203α,6β,7α-Trihydroxy-5β-cholanoic acid (α-muricholic acid)4536565769NDNDNDNDND32.263α,7α,12α-Trihydroxy-5β-cholanoic acid (cholic acid)12713715818113243627018811732.293α,6α-Dihydroxy-5β-cholanoic acid (hyodeoxycholic acid)60106129112114696471595132.45Unknown bile acid132024202010787832.513α,7β-Dihydroxy-5β-cholanoic acid (ursodeoxycholic acid)NDND11NDND6ND14NDND32.603β,12α-Dihydroxy-5α-cholanoic acid1216172120NDNDNDNDND32.843α,7α,12α-Trihydroxy-5β-homocholanoic acid (homocholic acid)8ND141510ND4NDNDND33.0612-Oxo-3α-hydroxy-5β-cholanoic acid6544857561263530232633.223α,6β,7β-Trihydroxy-5β-cholanoic acid (β-muricholic acid)76667714181455453717533.797-Oxo-3α,12α-dihydroxy-5β-cholanoic acid11ND9166ND566534.303α,6α,7β-Trihydroxy-5β-cholanoic acid (ω-muricholic acid)117140157194136393429293534.553α,6α,7β-Trihydroxy-5α-cholanoic acid (allo-ω-muricholic acid)273341433467466Total bile acids854955119413601116304363390414378a Bile acids are listed based on their retention times as methyl ester-trimethylsilyl ethers relative to a homologous series of n-alkanes, referred to as the methylene unit (MU) value.b Chemical structures were determined by electron ionization-gas chromatography-mass spectrometry.c ND, not detected. Open table in a new tab Several conclusions were reached from the data summarized in Table III., Table IV.. First, Cyp7−/− animals that survive to adulthood when maintained on unsupplemented chow have normal concentrations of total bile acids in their bile (Table III), but reduced concentrations of bile acids in their stool (Table IV). Second, the composition of bile in terms of the individual bile acid species is different in the bile and stool of wild-type and Cyp7−/− animals. Third, adult animals deficient in 7α-hydroxylase nevertheless synthesize and secrete 7α-hydroxylated bile acids into their bile and excrete these compounds in their stool. Bile acids with a 7α-hydroxyl group could arise in the mutant mice as a consequence of 7α-hydroxylase activity present in the intestinal flora or from the activity of another endogenous sterol 7α-hydroxylase enzyme. The data of Fig. 3 (lanes 1 and 3) demonstrate the presence of an enzyme activity in the livers of both wild-type and Cyp7−/− mice that is capable of 7α-hydroxylating cholest-5-ene-3β,25-diol (25-hydroxycholesterol) to form cholest-5-ene-3β,7α,25-triol. This oxysterol 7α-hydroxylase enzyme required NADPH as a cofactor (lanes 2 and 4). The specific activity of the enzyme was 6 pmol of product formed per min/mg of liver microsomal protein in adult wild-type mice and 5 pmol/min/mg of protein in Cyp7−/− mice. The experiment shown in Fig. 4 was performed to confirm that the product of this enzyme activity was cholest-5-ene-3β,7α,25-triol. An authentic standard representing the predicted product was chemically synthesized as described under “Experimental Procedures.” This standard was chromatographed on a thin-layer plate either separately (lane 1) or together (lanes 2 and 3) with the radiolabeled products derived by incubating liver extracts with [3H]cholest-5-ene-3β,25-diol. A comparison of the position of the radiolabeled product on the chromatogram (determined by autoradiography) to that of the standard (determined by phosphomolybdic acid staining) revealed that the two sterols comigrated in this solvent system. Similar results were obtained when product analysis was carried out in two other solvent systems (chloroform/methanol/H2O/ethyl acetate (50:20:4:15) and toluene/ethyl acetate (2:3)) on silica gel plates and when the sample was analyzed by chromatography on C18 plates using an acetonitrile/tetrahydrofuran (6:4) solvent system. Finally, analysis of the product by mass spectrometry confirmed the structure of the molecule as cholest-5-ene-3β,7α,25-triol (data not shown). We next determined the ability of different sterols to inhibit the oxysterol 7α-hydroxylase enzyme activity detected in the mutant mice. As shown by the data of Fig. 5, the product of the enzyme, cholest-5-ene-3β,7α,25-triol," @default.
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