24-hydroxycholesterol is a substrate for hepatic cholesterol 7alpha-hydroxylase (CYP7A)

(24S)-Hydroxycholesterol is formed from cholesterol in the brain and is important for cholesterol homeostasis in this organ. Elimination of (24S)-hydroxycholesterol has been suggested to occur in the liver but little is known about the metabolism of this oxysterol. In the present investigation, we report formation of 7alpha, 24-dihydroxycholesterol in pig and human liver. 7alpha-hydroxylase activity toward both isomers of 24-hydroxycholesterol [(24S) and (24R)] was found in a partially purified and reconstituted cholesterol 7alpha-hydroxylase (CYP7A) enzyme fraction from pig liver microsomes. In contrast, a purified enzyme fraction of pig liver oxysterol 7alpha-hydroxylase with high activity toward 27-hydroxycholesterol did not show any detectable activity toward 24-hydroxycholesterol. 7alpha-Hydroxylation of 24-hydroxycholesterol was strongly inhibited by 7-oxocholesterol, a known inhibitor of CYP7A. Human CYP7A, recombinantly expressed in Escherichia coli and in simian COS cells, showed 7alpha-hydroxylase activity toward both cholesterol and the two isomers of 24-hydroxycholesterol, with a preference for the (24S)-isomer. Our results show that 24-hydroxycholesterol is metabolized by CYP7A, an enzyme previously considered to be specific for cholesterol and cholestanol and not active toward oxysterols. Because CYP7A is the rate-limiting enzyme in the major pathway of bile acid biosynthesis, the possibility is discussed that at least part of the 24-hydroxycholesterol is converted into 7alpha-hydroxylated bile acids by the enzymes involved in the normal biosynthesis of bile acids.     

Differential expression of cholesterol hydroxylases in Alzheimer's disease

Cholesterol is eliminated from neurons by oxidization, which generates oxysterols. Cholesterol oxidation is mediated by the enzymes cholesterol 24-hydroxylase (CYP46A1) and cholesterol 27-hydroxylase (CYP27A1). Immunocytochemical studies show that CYP46A1 and CYP27A1 are expressed in neurons and some astrocytes in the normal brain, and CYP27A1 is present in oligodendrocytes. In Alzheimer's disease (AD), CYP46A1 shows prominent expression in astrocytes and around amyloid plaques, whereas CYP27A1 expression decreases in neurons and is not apparent around amyloid plaques but increases in oligodendrocytes. Although previous studies have examined the effects of synthetic oxysterols on the processing of amyloid precursor protein (APP), the actions of the naturally occurring oxysterols have yet to be examined. To understand the role of cholesterol oxidation in AD, we compared the effects of 24(S)- and 27-hydroxycholesterol on the processing of APP and analyzed the cell-specific expression patterns of the two cholesterol hydroxylases in the human brain. Both oxysterols inhibited production of Abeta in neurons, but 24(S)-hydroxycholesterol was approximately 1000-fold more potent than 27-hydroxycholesterol. The IC(50) of 24(S)-hydroxycholesterol for inhibiting Abeta secretion was approximately 1 nm. Both oxysterols induced ABCA1 expression with IC(50) values similar to that for inhibition of A beta secretion, suggesting the involvement of liver X receptor. Oxysterols also inhibited protein kinase C activity and APP secretion following stimulation of protein kinase C. The selective expression of CYP46A1 around neuritic plaques and the potent inhibition of APP processing in neurons by 24(S)-hydroxycholesterol suggests that CYP46A1 affects the pathophysiology of AD and provides insight into how polymorphisms in the CYP46A1 gene might influence the pathophysiology of this prevalent disease.     

Removal of cholesterol from extrahepatic sources by oxidative mechanisms.

Sterol 27-hydroxylase is an evolutionarily old cytochrome P450 species that is critical for oxidation of the side chain of cholesterol in connection with bile acid biosynthesis in the liver. The wide tissue and organ distribution of the enzyme suggests that it may also have other functions. It was recently shown that some cells (e.g. macrophages) have a high capacity to convert cholesterol into both 27-hydroxycholesterol and cholestenoic acid and that there is a significant flux of these steroids from extrahepatic sources to the liver where they are further oxidized into bile acids. The magnitude of this flux is such that it may be of importance for overall homeostasis of cholesterol. Very recently it was shown that the brain utilizes a similar mechanism for removal of cholesterol. A unique brain-specific 24S-hydroxylase converts cholesterol into 24S-hydroxycholesterol that is transported over the blood-brain barrier much more rapidly than unmetabolized cholestero. When 24S-hydroxycholesterol has reached the circulation it is taken up by the liver and further metabolized, most probably into bile acids. This flux is likely to be of importance for cholesterol homeostasis in the brain. This review summarizes our current knowledge regarding oxidative mechanisms for removal of extrahepatic cholesterol. It is evident that some cells utilize these mechanisms as alternatives or complements to the classical HDL-dependent reverse cholesterol transport.     

Cholesterol, hydroxycholesterols, and bile acids

Although a variety of oxidation products of cholesterol occur in vitro, enzyme-catalyzed oxidations can occur at only 5 sites on the cholesterol molecule: C7alpha, C22R, C24S, C25, and C27. The genes coding for the synthesis of these enzymes were cloned, the tissue expressions of the mRNAs were identified, and the enzymes were characterized. The biologic properties of the hydroxycholesterol molecules that are initially generated and their metabolites are under study. Downregulation of cholesterol synthesis via the SREBP/SCAP regulatory pathway is common to the initial hydroxycholesterols, but more variations exist with respect to these intermediates functioning as ligands for the nuclear receptor LXRalpha. Because this receptor regulates the expression of cholesterol 7alpha-hydroxylase and ABC transporter proteins, hydroxycholesterols and their intermediate steroid metabolites modulate a number of biologic processes. Metabolism of 22S-hydroxycholesterol to steroid hormones differs from that of the other hydroxycholesterols which form mostly steroid acidic products, otherwise known as bile acids. In vivo estimates of their production rates in intact humans indicate that 24S and 25-hydroxycholesterol account for no more than 7% of total bile acid production per day. Current evidence indicates that cholesterol 7alpha-hydroxycholesterol generated in the liver is the major source of bile acids in older adults. It is also known that the cholesterol 27-hydroxylation pathway is the only one expressed in fetal and neonatal life. Precisely when the proportions contributed by these two metabolic pathways to bile acid synthesis begin to shift and the role of the cholesterol 27-hydroxylase pathway in reverse cholesterol transport mandate further study.     

Transport of cholesterol into mitochondria is rate-limiting for bile acid synthesis via the alternative pathway in primary rat hepatocytes

Bile acid synthesis occurs mainly via two pathways: the "classic" pathway, initiated by microsomal cholesterol 7alpha-hydroxylase (CYP7A1), and an "alternative" (acidic) pathway, initiated by sterol 27-hydroxylase (CYP27). CYP27 is located in the inner mitochondrial membrane, where cholesterol content is very low. We hypothesized that cholesterol transport into mitochondria may be rate-limiting for bile acid synthesis via the "alternative" pathway. Overexpression of the gene encoding steroidogenic acute regulatory (StAR) protein, a known mitochondrial cholesterol transport protein, led to a 5-fold increase in bile acid synthesis. An increase in StAR protein coincided with an increase in bile acid synthesis. CYP27 overexpression increased bile acid synthesis by <2-fold. The rates of bile acid synthesis following a combination of StAR plus CYP27 overexpression were similar to those obtained with StAR alone. TLC analysis of (14)C-labeled bile acids synthesized in cells overexpressing StAR showed a 5-fold increase in muricholic acid; in chloroform-extractable products, a dramatic increase was seen in bile acid biosynthesis intermediates (27- and 7,27-hydroxycholesterol). High-performance liquid chromatography analysis showed that 27-hydroxycholesterol accumulated in the mitochondria of StAR-overexpressing cells only. These findings suggest that cholesterol delivery to the inner mitochondrial membrane is the predominant rate-determining step for bile acid synthesis via the alternative pathway.     

Regulation of sterol 27-hydroxylase and an alternative pathway of bile acid biosynthesis in primary cultures of rat hepatocytes

In man, hepatic mitochondrial sterol 27-hydroxylase and microsomal cholesterol 7-hydroxylase initiate distinct pathways of bile acid biosynthesis from cholesterol, the “acidic” and “neutral” pathways, respectively. A similar acidic pathway in the rat has been hypothesized, but its quantitative importance and ability to be regulated at the level of sterol 27-hydroxylase are uncertain. In this study, we explored the molecular regulation of sterol 27-hydroxylase and the acidic pathway of bile acid biosynthesis in primary cultures of adult rat hepatocytes. mRNA and protein turnover rates were approximately 10-fold slower for sterol 27-hydroxylase than for cholesterol 7-hydroxylase. Sterol 27-hydroxylase mRNA was not spontaneously expressed in culture. The sole requirement for preserving sterol 27-hydroxylase mRNA at the level of freshly isolated hepatocytes (0 h) after 72 h was the addition of dexamethasone (0.1 μM; > 7-fold induction). Sterol 27-hydroxylase mRNA, mass and specific activity were not affected by thyroxine (1.0 μM), dibutyryl-cAMP (50 μM), nor squalestatin 1 (150 nM-1.0 μM), an inhibitor of cholesterol biosynthesis. Taurocholate (50 μM), however, repressed sterol 27-hydroxylase mRNA levels by 55%. Sterol 27-hydroxylase specific activity in isolated mitochondria was increased > 10-fold by the addition of 2-hydroxypropyl-β-cyclodextrin. Under culture conditions designed to maximally repress cholesterol 7-hydroxylase and bile acid synthesis from the neutral pathway but maintain sterol 27-hydroxylase mRNA and activity near 0 h levels, bile acid synthesis from [¹⁴C]cholesterol remained relatively high and consisted of β-muricholate, the product of chenodeoxycholate in the rat. We conclude that rat liver harbors a quantitatively important alternative pathway of bile acid biosynthesis and that its initiating enzyme, sterol 27-hydroxylase, may be slowly regulated by glucocorticoids and bile acids.     

Oxysterol 7 alpha-hydroxylase activity by cholesterol 7 alpha-hydroxylase (CYP7A)

A 7 alpha-hydroxylation is necessary for conversion of both cholesterol and 27-hydroxycholesterol into bile acids. According to current theories, cholesterol 7 alpha-hydroxylase (CYP7A) is responsible for the former and oxysterol 7 alpha-hydroxylase (CYP7B) for the latter reaction. CYP7A is believed to have a very high substrate specificity whereas CYP7B is active toward oxysterols, dehydroepiandrosterone, and pregnenolone. In the present study, 7 alpha-hydroxylation of various oxysterols in liver and kidney was investigated. Surprisingly, human cholesterol 7 alpha-hydroxylase, CYP7A, expressed as a recombinant in Escherichia coli and COS cells, was active toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol. This enzyme has previously been thought to be specific for cholesterol and cholestanol. A partially purified and reconstituted cholesterol 7 alpha-hydroxylase enzyme fraction from pig liver showed 7 alpha-hydroxylase activity toward the same oxysterols as metabolized by expressed recombinant human and rat CYP7A. The 7 alpha-hydroxylase activity toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol in rat liver was significantly increased by treatment with cholestyramine, an inducer of CYP7A. From the present results it may be concluded that CYP7A is able to function as an oxysterol 7 alpha-hydroxylase, in addition to the previously known human oxysterol 7 alpha-hydroxylase, CYP7B. These findings may have implications for oxysterol-mediated regulation of gene expression and for pathways of bile acid biosynthesis. A possible use of 20(S)-hydroxycholesterol as a marker substrate for CYP7A is proposed.     

Synthesis and pharmacokinetic study of a 11C-labeled cholesterol 24-hydroxylase inhibitor using ‘in-loop’ [11C]CO2 fixation method

Cholesterol 24-hydroxylase, also known as CYP46A1 (EC 1.14.13.98), is a monooxygenase and a member of the cytochrome P450 family. CYP46A1 is specifically expressed in the brain where it controls cholesterol elimination by producing 24S-hydroxylcholesterol (24-HC) as the major metabolite. Modulation of CYP46A1 activity may affect Aβ deposition and p-tau accumulation by changing 24-HC formation, which thereafter serves as potential therapeutic pathway for Alzheimer’s disease. In this work, we showcase the efficient synthesis and preliminary pharmacokinetic evaluation of a novel cholesterol 24-hydroxylase inhibitor 1 for use in positron emission tomography.     

26-Hydroxycholesterol: synthesis, metabolism, and biologic activities

Cholest-5-ene-3 beta,26-diol (26-hydroxycholesterol) is synthesized by a mitochondrial P-450 enzyme that appears to be widely distributed in tissues. Together with other C-27 steroid intermediates it is transported to the liver and metabolized to bile acids. Although 26-hydroxycholesterol is transported in plasma lipoproteins mostly as the fatty acid ester, neither its assembly and orientation within lipoproteins nor its mechanism of transport across the sinusoidal liver membrane is known. Cell culture studies indicate that 26-hydroxycholesterol can inhibit both cholesterol synthesis and low density lipoprotein (LDL) receptor activity. Inhibition of DNA synthesis also occurs and may not be related to the reduction in HMG-CoA reductase activity. The relationship of these in vitro activities to the physiologic role(s) of 26-hydroxycholesterol remains to be clarified. A clue to its biologic role is the knowledge that markedly decreased 26-hydroxylase activity appears to be the molecular basis of cerebrotendinous xanthomatosis, an inborn error of metabolism characterized by a significant decrease in 26-hydroxycholesterol and bile acid synthesis and an increase in cholesterol synthesis.     

Alternate pathways of bile acid synthesis in the cholesterol 7alpha-hydroxylase knockout mouse are not upregulated by either cholesterol or cholestyramine feeding

Bile acids are synthesized via the classic pathway initiated by cholesterol 7alpha-hydroxylase (CYP7A1), and via alternate pathways, one of which is initiated by sterol 27-hydroxylase (CYP27). These studies used mice lacking cholesterol 7alpha-hydroxylase (Cyp7a1(-/-)) to establish whether the loss of the classic pathway affected cholesterol homeostasis differently in males and females, and to determine if the rate of bile acid synthesis via alternate pathways was responsive to changes in the enterohepatic flux of cholesterol and bile acids. In both the Cyp7a1(-/-) males and females, the basal rate of bile acid synthesis was only half of that in matching Cyp7a1(+/+) animals. Although bile acid pool size contracted markedly in all the Cyp7a1(-/-) mice, the female Cyp7a1(-/-) mice maintained a larger, more cholic acid-rich pool than their male counterparts. Intestinal cholesterol absorption in the Cyp7a1(-/-) males fell from 46% to 3%, and in the matching females from 58% to 17%. Bile acid synthesis in Cyp7a1(+/+) males and females was increased 2-fold by cholesterol feeding, and 4-fold by cholestyramine treatment, but was not changed in matching Cyp7a1(-/-) mice by either of these manipulations. In the Cyp7a1(-/-) mice fed cholesterol, hepatic cholesterol concentrations increased only marginally in the males, but rose almost 3-fold in the females. CYP7A1 activity and mRNA levels were greater in females than in males, and were increased by cholesterol feeding in both sexes. CYP27 activity and mRNA levels did not vary as a function of CYP7A1 genotype, gender, or dietary cholesterol intake. We conclude that in the mouse the rate of bile acid synthesis via alternative pathways is unresponsive to changes in the enterohepatic flux of cholesterol and bile acid, and that factors governing gender-related differences in bile acid synthesis, pool size, and pool composition play an important role in determining the impact of CYP7A1 deficiency on cholesterol homeostasis in this species.     

Quantitation of two pathways for cholesterol excretion from the brain in normal mice and mice with neurodegeneration

Although the pool of cholesterol in the adult central nervous system (CNS) is large and of constant size, little is known of the process(es) involved in regulation of sterol turnover in this pool. In 7-week-old mice, net excretion of cholesterol from the brain equaled 1.4 mg/day/kg body weight, and from the whole animal was 179 mg/day/kg. Deletion of cholesterol 24-hydroxylase, an enzyme highly expressed in the CNS, did not alter brain growth or myelination, but reduced sterol excretion from the CNS 64% to 0.5 mg/day/kg. In mice with a mutation in the Niemann-Pick C gene that had ongoing neurodegeneration, sterol excretion from the CNS was increased to 2.3 mg/day/kg. Deletion of cholesterol 24-hydroxylase activity in these animals reduced net excretion only 22% to 1.8 mg/day/kg. Thus, at least two different pathways promote net sterol excretion from the CNS. One uses cholesterol 24-hydroxylase and may reflect sterol turnover in large neurons in the brain. The other probably involves the movement of cholesterol or one of its metabolites across the blood-brain barrier and may more closely mirror sterol turnover in pools such as glial cell membranes and myelin.     

Human sterol 27-hydroxylase (CYP27) overexpressor transgenic mouse model. Evidence against 27-hydroxycholesterol as a critical regulator of cholesterol homeostasis

CYP27-overexpressed transgenic mice were generated with the use of a human full-length CYP27 coding region cloned into a ubiquitous expression vector. Positive transgenic mice were identified by tail DNA genotyping and high fecal 27-hydroxycholesterol content. The levels of 27-hydroxycholesterol were found to be 3-5 times higher in the circulation and the tissues of the overexpressed mice when compared with littermate controls. There were no gross morphological differences between the overexpressed mice and their controls. Total cholesterol and triglyceride levels were not affected by overexpression of CYP27. Serum lathosterol was also normal, suggesting a normal rate of cholesterol synthesis. Serum levels of 7alpha-hydroxycholesterol were unaffected, suggesting a normal rate of bile acid formation in the pathway involving cholesterol 7alpha-hydroxylase. Biliary bile acid composition was slightly affected by CYP27 overexpression in female but not in male mice. Fecal levels of neutral steroids were slightly but significantly increased in overexpressor female mice but not in male mice. Levels of 24-hydroxycholesterol in the circulation were significantly reduced in the overexpressed mice, probably as a consequence of a recently described catabolic pathway involving CYP27. Combined with the results of our previous work on mice with a disruption of the CYP27 gene, the present results suggest that the levels of 27-hydroxycholesterol are not of critical importance for cholesterol homeostasis in mice.     

24S-hydroxycholesterol in plasma: A marker of cholesterol turnover in neurodegenerative diseases

Brain cholesterol is mainly involved in the cell membrane structure, in signal transduction, neurotransmitter release, synaptogenesis and membrane trafficking. Impairment of brain cholesterol metabolism was described in neurodegenerative diseases, such as Multiple Sclerosis, Alzheimer and Huntington Diseases. Since the blood–brain barrier efficiently prevents cholesterol uptake from the circulation into the brain, de novo synthesis is responsible for almost all cholesterol present there. Cholesterol is converted into 24S-hydroxycholesterol (24OHC) by cholesterol 24-hydroxylase (CYP46A1) expressed in neural cells.     

Expression cloning of an oxysterol 7alpha-hydroxylase selective for 24-hydroxycholesterol

The synthesis of 7alpha-hydroxylated bile acids from oxysterols requires an oxysterol 7alpha-hydroxylase encoded by the Cyp7b1 locus. As expected, mice deficient in this enzyme have elevated plasma and tissue levels of 25- and 27-hydroxycholesterol; however, levels of another major oxysterol, 24-hydroxycholesterol, are not increased in these mice, suggesting the presence of another oxysterol 7alpha-hydroxylase. Here, we describe the cloning and characterization of murine and human cDNAs and genes that encode a second oxysterol 7alpha-hydroxylase. The genes contain 12 exons and are located on chromosome 6 in the human (CYP39A1 locus) and in a syntenic position on chromosome 17 in the mouse (Cyp39a1 locus). CYP39A1 is a microsomal cytochrome P450 enzyme that has preference for 24-hydroxycholesterol and is expressed in the liver. The levels of hepatic CYP39A1 mRNA do not change in response to dietary cholesterol, bile acids, or a bile acid-binding resin, unlike those encoding other sterol 7alpha-hydroxylases. Hepatic CYP39A1 expression is sexually dimorphic (female > male), which is opposite that of CYP7B1 (male > female). We conclude that oxysterol 7alpha-hydroxylases with different substrate specificities exist in mice and humans and that sexually dimorphic expression patterns of these enzymes in the mouse may underlie differences in bile acid metabolism between the sexes.     

Decreased content,rate of synthesis and export of cholesterol in the brain of apoE knockout mice

Apolipoprotein E knockout (apoE-KO) mice present synaptic loss, cognitive dysfunction, and high plasma lipid levels that may affect brain function simulating Alzheimer disease. Plasma and brain sterols were measured in apoE-KO and in wild type control mice on a cholesterol-free, phytosterol-containing diet by gas chromatography coupled to a mass spectrometer. Plasma cholesterol and phytosterols (campesterol and sitosterol) were higher in apoE-KO compared to control mice. Cholesterol precursors (desmosterol and lathosterol) were not detected in plasma of control mice but were present in apoE-KO mice. In the brain amounts of cholesterol, desmosterol, campesterol and 24-hydroxycholesterol were significantly lower in apoE-KO than in controls. There is a tendency in apoE-KO for lower values of 7α-hydroxycholesterol and 7β-hydroxycholesterol. Cholesterol content, synthesis rates (desmosterol) and export of 24-hydroxycholesterol are reduced in the brain of the severe hypercholesterolemic apoE-KO mice.     

Cytochrome P450s in the synthesis of cholesterol and bile acids--from mouse models to human diseases

The present review describes the transgenic mouse models that have been designed to evaluate the functions of the cytochrome P450s involved in cholesterol and bile acid synthesis, as well as their link with disease. The knockout of cholesterogenic Cyp51 is embrionally lethal, with symptoms of Antley-Bixler syndrome occurring in mice, whereas the evidence for this association is conflicting in humans. Disruption of Cyp7a1 from classic bile acid synthesis in mice leads to either increased postnatal death or a milder phenotype with elevated serum cholesterol. The latter is similar to the case in humans, where CYP7A1 mutations associate with high plasma low-density lipoprotein and hepatic cholesterol content, as well as deficient bile acid excretion. Disruption of Cyp8b1 from an alternative bile acid pathway results in the absence of cholic acid and a reduced absorption of dietary lipids; however, the human CYP8B1 polymorphism fails to explain differences in bile acid composition. Unexpectedly, apparently normal Cyp27a1(-/-) mice still synthesize bile acids that originate from the compensatory pathway. In humans, CYP27A1 mutations cause cerebrotendinous xanthomatosis, suggesting that only mice can compensate for the loss of alternative bile acid synthesis. In line with this, Cyp7b1 knockouts are also apparently normal, whereas human CYP7B1 mutations lead to a congenital bile acid synthesis defect in children or spastic paraplegia in adults. Mouse knockouts of the brain-specific Cyp46a1 have reduced brain cholesterol excretion, whereas, in humans, CYP46A1 polymorphisms associate with cognitive impairment. At present, cytochrome P450 family 39 is poorly characterized. Despite important physiological differences between humans and mice, mouse models prove to be an invaluable tool for understanding the multifactorial facets of cholesterol and bile acid-related disorders.     

Bile acid synthesis in the Smith-Lemli-Opitz syndrome: effects of dehydrocholesterols on cholesterol 7alpha-hydroxylase and 27-hydroxylase activities in rat liver.

The Smith-Lemli-Opitz syndrome (SLOS) is a congenital birth defect syndrome caused by a deficiency of 3beta-hydroxysterol Delta(7)-reductase, the final enzyme in the cholesterol biosynthetic pathway. The patients have reduced plasma and tissue cholesterol concentrations with the accumulation of 7-dehydrocholesterol and 8-dehydrocholesterol. Bile acid synthesis is reduced and unnatural cholenoic and cholestenoic acids have been identified in some SLOS patients. To explore the mechanism of the abnormal bile acid production, the activities of key enzymes in classic and alternative bile acid biosynthetic pathways (microsomal cholesterol 7alpha-hydroxylase and mitochondrial sterol 27-hydroxylase) were measured in liver biopsy specimens from two mildly affected SLOS patients. The effects of 7- and 8-dehydrocholesterols on these two enzyme activities were studied by using liver from SLOS model rats that were treated with the Delta(7)-reductase inhibitor (BM15.766) for 4 months and were comparable with more severe SLOS phenotype in plasma and hepatic sterol compositions. In the SLOS patients, cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase were not defective. In BM15.766-treated rats, both enzyme activities were lower than those in control rats and they were competitively inhibited by 7- and 8-dehydrocholesterols. Rat microsomal cholesterol 7alpha-hydroxylase did not transform 7-dehydrocholesterol or 8-dehydrocholesterol into 7alpha-hydroxylated sterols. In contrast, rat mitochondrial sterol 27-hydroxylase catalyzed 27-hydroxylation of 7- and 8-dehydrocholesterols, which were partially converted to 3beta-hydroxycholestadienoic acids. Addition of microsomes to the mitochondrial 27-hydroxylase assay mixture reduced 27-hydroxydehydrocholesterol concentrations, which suggested that 27-hydroxydehydrocholesterols were further metabolized by microsomal enzymes. These results suggest that reduced normal bile acid production is characteristic of severe SLOS phenotype and is caused not only by depletion of hepatic cholesterol but also by competitive inhibition of cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase activities by accumulated 7- and 8-dehydrocholesterols. Unnatural bile acids are synthesized mainly by the alternative pathway via mitochondrial sterol 27-hydroxylase in SLOS.     

Disruption of the oxysterol 7alpha-hydroxylase gene in mice

Mice without oxysterol 7alpha-hydroxylase, an enzyme of the alternate bile acid synthesis pathway with a sexually dimorphic expression pattern, were constructed by the introduction of a null mutation at the Cyp7b1 locus. Animals heterozygous (Cyp7b1(+/-)) and homozygous (Cyp7b1(-/-)) for this mutation were grossly indistinguishable from wild-type mice. Plasma and tissue levels of 25- and 27-hydroxycholesterol, two oxysterol substrates of this enzyme with potent regulatory actions in cultured cells, were markedly elevated in Cyp7b1(-/-) knockout animals. Parameters of bile acid metabolism as well as plasma cholesterol and triglyceride levels in male and female Cyp7b1(-/-) mice were normal. The cholesterol contents of major tissues were not altered. In vivo sterol biosynthetic rates were unaffected in multiple tissues with the exception of the male kidney, which showed a approximately 40% decrease in de novo synthesis versus controls. We conclude that the major physiological role of the CYP7B1 oxysterol 7alpha-hydroxylase is to metabolize 25- and 27-hydroxycholesterol and that loss of this enzyme in the liver is compensated for by increases in the synthesis of bile acids by other pathways. A failure to catabolize oxysterols in the male kidney may lead to a decrease in de novo sterol synthesis.