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.

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 7␣-hydroxycholesterol was less than 15% of normal. Compensatory up-regulation of hepatic cholesterol 7␣-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 the cyp27 ؊/؊ 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.

HIV produces a chronic viral infection of the central nervous system that elicits chronic glial activation and overexpression of glial cytokines that are also implicated in Alzheimer disease (AD) pathogenesis. A genetic risk factor for AD is the E4 isoform for apolipoprotein E (APOE). Here we compare the frequency of neurologic symptoms for subjects with and without the E4 isoform (E4(+)and E4(-), respectively) in an HIV cohort. Compared with E4(-) subjects, twice as many E4(+) subjects were demented (30% compared with 15%) or had peripheral neuropathy (70% compared with 39%) at least once, and they had threefold more symptomatic examinations (13% compared with 3% and 42% compared with 14%, respectively)(P < 0.0001). Thus, neurologic symptoms for HIV-infection and AD are linked through an etiologic risk factor. Long-term survivors of HIV infection with E4 may be at high risk for AD; conversely, gene-viral interactions may speed AD pathogenesis.

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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 ...