One of the major oxysterols in the human circulation is 4␤-hydroxycholesterol formed from cholesterol by the drug-metabolizing enzyme cytochrome P450 3A4. Deuterium-labeled 4␤-hydroxycholesterol was injected into two healthy volunteers, and the apparent half-life was found to be 64 and 60 h, respectively. We have determined earlier the half-lives for 7␣-, 27-, and 24-hydroxycholesterol to be ϳ0.5, 0.75, and 14 h, respectively. Patients treated with certain antiepileptic drugs have up to 20-fold increased plasma concentrations of 4␤-hydroxycholesterol. The apparent half-life of deuteriumlabeled 4␤-hydroxycholesterol in such a patient was found to be 52 h, suggesting that the high plasma concentration was because of increased synthesis rather than impaired clearance. 4␤-Hydroxycholesterol was converted into acidic products at a much slower rate than 7␣-hydroxycholesterol in primary human hepatocytes, and 4␤-hydroxycholesterol was 7␣-hydroxylated at a slower rate than cholesterol by recombinant human CYP7A1. CYP7B1 and CYP39A1 had no activity toward 4␤-hydroxycholesterol. These results suggest that the high plasma concentration of 4␤-hydroxycholesterol is because of its exceptionally slow elimination, probably in part because of the low rate of 7␣-hydroxylation of the steroid. The findings are discussed in relation to a potential role of 4␤-hydroxycholesterol as a ligand for the nuclear receptor LXR.4␤-Hydroxycholesterol is one of the quantitatively most important oxysterols in human circulation (1). We have recently shown that it is formed by the drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) 1 (1). Preliminary experiments showed that the formation of this oxysterol by human liver microsomes was relatively slow. The high plasma levels of the oxysterol are therefore surprising, and we hypothesized that this may be a consequence of slow metabolism. Therefore, in this work, we determined the rate of elimination of deuteriumlabeled 4␤-hydroxycholesterol from plasma. Oxysterols are generally degraded to bile acids, and the rate-limiting step in this conversion is the introduction of a hydroxyl group in the 7␣-position of the steroid. Alternative pathways for bile acid biosynthesis start with oxidation of the steroid side chain by CYP27A1 and CYP46. Therefore, we have studied the possibility that these cytochromes are active toward 4␤-hydroxycholesterol. The metabolism of 4␤-hydroxycholesterol was studied in human primary hepatocytes, control, and transfected cells and by incubations with recombinant enzymes. In addition, fecal samples from three untreated subjects and one subject treated with carbamazepine were analyzed for 4␤-hydroxylated bile acids. Based on these experiments, we present evidence that 4␤-hydroxycholesterol has an unusually long halflife in plasma and that this is the result of slow elimination, particularly slow 7␣-hydroxylation that is the rate-limiting step for further conversion into bile acids.

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Mammalian CNS contains a disproportionally large and remarkably stable pool of cholesterol. Despite an efficient recycling there is some requirement for elimination of brain cholesterol. Conversion of cholesterol into 24S-hydroxycholesterol by the cholesterol 24-hydroxylase (CYP46A1) is the quantitatively most important mechanism. Based on the protein expression and plasma levels of 24S-hydroxycholesterol, CYP46A1 activity appears to be highly stable in adults. Here we have made a structural and functional characterization of the promoter of the human CYP46A1 gene. No canonical TATA or CAAT boxes were found in the promoter region. Moreover this region had a high GC content, a feature often found in genes considered to have a largely housekeeping function. A broad spectrum of regulatory axes using a variety of promoter constructs did not result in a significant transcriptional regulation. Oxidative stress caused a significant increase in transcriptional activity. The possibility of a substrate-dependent transcriptional regulation was explored in vivo in a sterol-deficient mouse model (Dhcr24 null) in which almost all cholesterol had been replaced with desmosterol, which is not a substrate for CYP46A1. Compared with heterozygous littermates there was no statistically significant difference in the mRNA levels of Cyp46a1. During the first 2 weeks of life in the wild-type mouse, however, a significant increase of Cyp46a1 mRNA levels was found, in parallel with an increase in 24S-hydroxycholesterol level and a reduction of cholesterol synthesis. The failure to demonstrate a significant transcriptional regulation under most conditions is discussed in relation to the turnover of brain and neuronal cholesterol.Although the brain is the most cholesterol-rich organ in the body, relatively little is known about the mechanisms by which it maintains steady-state cholesterol levels (1, 2). This is in marked contrast to the situation in virtually every other tissue or organ. One finding that has been consistently confirmed is that, due to the efficiency of the bloodbrain barrier, the brain is unable to take up cholesterol from the circulation and relies on de novo synthesis to meet its substantial cholesterol requirements. However, the rate of cholesterol synthesis in the adult brain is very low, and the bulk of brain cholesterol has a half-life that is at least 100 times longer than that of cholesterol in most other organs (3).One consequence of this "uncoupling" of brain and whole body cholesterol homeostasis has been the evolution of specific mechanisms for maintenance of cerebral cholesterol levels. Two mechanisms for removal of brain cholesterol are currently recognized (1). The first is analogous to classic "reverse cholesterol transport" and is mediated by a flux of cholesterol present in apolipoprotein E containing lipoproteins through cerebrospinal fluid into the circulation (4, 5). In adults, this mechanism is believed to be responsible for elimination of 1-2 mg of cholesterol per 24 h. The details of this particular ...

Recently, we demonstrated a net blood-to-brain passage of the oxysterol 27-hydroxycholesterol corresponding to 4-5 mg/day. As the steady-state levels of this sterol are only 1-2 mg/g brain tissue, we hypothesized that it is metabolized and subsequently eliminated from the brain. To explore this concept, we first measured the capacity of in vitro systems representing the major cell populations found in the brain to metabolize 27-hydroxycholesterol. We show here that 27-hydroxycholesterol is metabolized into the known C 27 steroidal acid 7a-hydroxy-3-oxo-4-cholestenoic acid by neuronal cell models only. Using an in vitro model of the blood-brain barrier, we demonstrate that 7a-hydroxy-3-oxo-4-cholestenoic acid is efficiently transferred across monolayers of primary brain microvascular endothelial cells. Finally, we measured the concentration of 7a-hydroxy-3-oxo-4-cholestenoic acid in plasma from the internal jugular vein and brachial artery of healthy volunteers. Calculation of the arteriovenous concentration difference revealed a significant in vivo flux of this steroid from the brain into the circulation in human.Together, these studies identify a novel metabolic route for the elimination of 27-hydroxylated sterols from the brain. Given the emerging connections between cholesterol and neurodegeneration, this pathway may be of importance for the development of these conditions.

On a global scale, there is an increasing tendency for a more aggressive treatment of hypercholesterolemia. Minor effects of statins on brain cholesterol metabolism have been reported in some in vivo animal studies, and it seems that this is due to a local effect of the drug. We treated male mice of the inbred strain C57/BL6 with a high daily dose of lipophilic simvastatin (100 mg/kg b.wt.) or hydrophilic pravastatin (200 mg/kg b.wt.) or vehicle (controls) by oral gavage for 3 days. To compare the impact of both statins on brain cholesterol synthesis and degradation, levels of cholesterol, its precursor lathosterol, and its brain metabolite 24(S)-hydroxycholesterol as well as statin concentrations were determined in whole-brain lipid extracts using mass spectrometry. The expression of 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase mRNA and of other target genes were evaluated using real-time reverse transcription-polymerase chain reaction. In addition, analysis of liver and serum samples was performed. Similar levels of simvastatin and pravastatin were detected in whole-brain homogenates. Cholesterol contents in the brain, liver, and serum were not affected by high-dose statin treatment. Whereas brain cholesterol precursor levels were reduced in simvastatin-treated animals only, no effect was observed on the formation of the brain cholesterol metabolite, 24(S)-hydroxycholesterol. Polymerase chain reaction analysis revealed that mRNA expression of HMG-CoA reductase and ATP-binding cassette transporter A1 in the brain was significantly up-regulated in simvastatintreated animals compared with pravastatin-treated or control animals. We conclude that, under the present experimental conditions, brain cholesterol synthesis is significantly affected by short-term treatment with high doses of lipophilic simvastatin, whereas whole-brain cholesterol turnover is not disturbed.