There is a continuous flux of the oxysterol 27-hydroxycholesterol (27-OHC) from the circulation across the blood-brain barrier (BBB) into the brain. The major metabolite of 27-OHC in the brain is 7α-hydroxy-3-oxo-4-cholestenoic acid (7-HOCA). We confirm a recent report describing the presence of this metabolite in cerebrospinal fluid (CSF) at a relatively high concentration. A simple and accurate method was developed for assay of 7-HOCA in CSF based on isotope dilution-mass spectrometry and use of 2H4-labeled internal standard. The concentration of this metabolite was found to be markedly increased in CSF from patients with a dysfunctional BBB. There was a high correlation between the levels of 7-HOCA in CSF and the CSF/serum albumin ratio. The concentration of 7-HOCA in CSF was not significantly affected by neurodegeneration. Our findings suggest that 7-HOCA could be used as a diagnostic marker for conditions with a dysfunctional BBB.

The synthetic inhibitors of sterol biosynthesis, 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one and 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one, are of interest as potential cholesterol lowering drugs. Rapid metabolism of synthetic 15-ketosterols may lead to a decrease, or loss, of their potency to affect lipid metabolism. 3beta-Hydroxy-5alpha-cholest-8(14)-en-15-one is reported to be rapidly side chain oxygenated by rat liver mitochondria. In an attempt to reduce this metabolism, the novel side chain modified 15-ketosterol 3beta-Hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one was synthesized. We have examined the metabolism by recombinant human CYP27A1 of this novel side chain modified 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one and compared the rate of metabolism with that of the previously described 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one. Both sterols were found to be efficiently metabolized by recombinant human CYP27A1. None of the two 15-ketosterols was significantly metabolized by microsomal 7alpha-hydroxylation. Interestingly, CYP27A1-mediated product formation was much lower with the side chain modified 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one than with the previously described 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one. A surprising finding was that this novel side chain modified sterol was metabolized mainly in the C-28 position by CYP27A1. The data on 28-hydroxylation by human CYP27A1 provide new insights on the catalytic properties and substrate specificity of this enzyme. The finding that 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one with a modified side chain is metabolized at a dramatically slower rate than the previously described 15-ketosterol with unmodified side chain may be important for future development of synthetic cholesterol lowering sterols.

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 mu g/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 7 alpha-hydroxy-3-oxo-4-cholestenoic acid by neuronal cell models only. Using an in vitro model of the blood-brain barrier, we demonstrate that 7 alpha-hydroxy-3-oxo-4-cholestenoic acid is efficiently transferred across monolayers of primary brain microvascular endothelial cells. Finally, we measured the concentration of 7 alpha-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.

The most serious consequence of sterol 27-hydroxylase deficiency in humans [cerebrotendinous xanthomatosis (CTX)] is the development of cholestanol-containing brain xanthomas. The cholestanol in the brain may be derived from the circulation or from 7a-hydroxylated intermediates in bile acid synthesis, present at 50- to 250-fold increased levels in plasma. Here, we demonstrate a transfer of 7 alpha-hydroxy-4-cholesten-3-one across cultured porcine brain endothelial cells (a model for the blood-brain barrier) that is similar to 100-fold more efficient than the transfer of cholestanol. Furthermore, there was an efficient conversion of 7 alpha-hydroxy-4-cholesten-3-one to cholestanol in cultured neuronal and glial cells as well as in monocyte-derived macrophages of human origin. It is concluded that the continuous intracellular production of cholestanol from a bile acid precursor capable of rapidly passing biomembranes, including the blood-brain barrier, is likely to be of major importance for the accumulation of cholestanol in patients with CTX. Such a mechanism also fits well with the observation that treatment with chenodeoxycholic acid, which normalizes the level of the bile acid precursor, results in a reduction of cholestanol-containing xanthomas even in the brain.

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.

A wide variety of endogenous carboxylic acids and xenobiotics are conjugated with amino acids, before excretion in urine or bile. The conjugation of carboxylic acids and bile acids with taurine and glycine has been widely characterized, and de novo synthesized bile acids are conjugated to either glycine or taurine in peroxisomes. Peroxisomes are also involved in the oxidation of several other lipid molecules, such as very long chain acyl-CoAs, branched chain acyl-CoAs, and prostaglandins. In this study, we have now identified a novel peroxisomal enzyme called acyl-coenzyme A:amino acid N-acyltransferase (ACNAT1). Recombinantly expressed ACNAT1 acts as an acyltransferase that efficiently conjugates very long-chain and long-chain fatty acids to taurine. The enzyme shows no conjugating activity with glycine, showing that it is a specific taurine conjugator. Acnat1 is mainly expressed in liver and kidney, and the gene is localized in a gene cluster, together with two further acyltransferases, one of which conjugates bile acids to glycine and taurine. In conclusion, these data describe ACNAT1 as a new acyltransferase, involved in taurine conjugation of fatty acids in peroxisomes, identifying a novel pathway for production of N-acyltaurines as signaling molecules or for excretion of fatty acids.

Sterol 12α-hydroxylase (CYP8B1) is an obligatory enzyme for the synthesis of cholic acid and regulation of liver bile acid synthesis and intestine cholesterol absorption. The present study evaluates the roles for sterol regulatory element binding proteins (SREBPs) in the regulation of the CYP8B1 gene. Cholesterol feeding of mice and rats decreased the activity of CYP8B1, contrary to the up-regulation of cholesterol 7α-hydroxylase (CYP7A1). Cholesterol feeding also reduced mRNA levels for SREBP-1 but not for SREBP-2 in rat livers. Cholesterol and 25-hydroxycholesterol decreased the CYP8B1/luciferase reporter activity. Co-transfection of SREBP-1a and -1c stimulated CYP8B1 promoter activity, while SREBP-2 did not have any effects. Electrophoretic mobility shift assay and mutagenesis analyses identified several functional sterol regulatory elements (SRE) and E-box motifs in the rat CYP8B1 promoter. Our results indicate that SREBP-1a and -1c enhance transcription of the CYP8B1 gene through binding to SRE. Cholesterol loading reduces SREBP-1 mRNA expression in addition to reducing functional SREBP-1 protein, and results in decreasing CYP8B1 gene transcription.

Recent epidemiological studies suggest that inhibitors of 3-hydroxy-3-methyl-glutaryl CoA reductase, so-called statins, are effective in lowering the prevalence of Alzheimer’s disease. Whether the effect of statins is due to a local inhibition of cholesterol synthesis in the brain or whether it is mediated by the reduced levels of cholesterol in the circulation is not known. In the present work, we tested the possibility that high doses of lipophilic and hydrophilic statins, simvastatin and pravastatin, respectively, or a diet high in cholesterol could affect cholesterol homeostasis in the brain of guinea pigs. The total brain cholesterol levels were not affected by high-dose simvastatin or pravastatin treatment. Significantly lower levels of the cholesterol precursor lathosterol and its ratio to cholesterol were found in the brains of simvastatin and pravastatin-treated animals. 24S-Hydroxycholesterol, the transportable form of cholesterol across the blood–brain barrier, was significantly lower in the brain of pravastatin-treated animals. Excessive cholesterol feeding resulted in higher serum cholesterol levels but did not affect total brain cholesterol level. However, de novo cholesterol synthesis in the brain seemed to be down-regulated, as indicated by lower absolute levels and cholesterol-related ratios of lathosterol compared with controls. The passage of deuterium-labeled cholesterol across the blood–brain barrier in one animal was found to be approximately 1%. Our results suggest that brain cholesterol synthesis in guinea pigs can be slightly, but significantly, influenced by high doses of lipophilic and hydrophilic statins as well as by high dietary cholesterol intake, while total brain cholesterol content and thus, cholesterol homeostasis is maintained.

The brain is the almost exclusive site of formation of 24S-hydroxycholesterol in man, and there is a continuous flux of this oxysterol across the blood-brain barrier into the circulation. The hepatic metabolism of 24S-hydroxycholesterol was studied here by three different approaches: incubation of tritium-labeled 24S-hydroxycholesterol with human primary hepatocytes, administration of tritium-labeled 24S-hydroxycholesterol to a human volunteer, and quantitation of free and conjugated 24S-hydroxycholesterol and its neutral metabolites in ileocecal fluid from patients with ileal fistulae. 24S-Hydroxycholesterol as well as 24R-hydroxycholesterol were converted into bile acids by human hepatocytes at a rate of about 40% of that of the normal intermediate in bile acid synthesis, 7α-hydroxycholesterol. There was also a conversion of 24S-hydroxycholesterol into conjugate(s) of 5-cholestene-3β,24S,27-triol at a rate similar to the that of conversion into bile acids. When administered to a human volunteer, labeled 24S-hydroxycholesterol was converted into bile acids at about half the rate of simultaneously administered labeled 7α-hydroxycholesterol. Free, sulfated, and glucuronidated 24S-hydroxycholesterol and 5-cholestene-3β,24,27-triol were identified in ileocecal fluid. The excretion of these steroids was about 3.5 mg/24 h, amounting to more than 50% of the total estimated flux of 24S-hydroxycholesterol from the brain. It is concluded that 24S-hydroxycholesterol is a less efficient precursor to bile acids and that about half of it is conjugated and eliminated in bile as such or as a conjugate of a 27-hydroxylated metabolite. The less efficient metabolism of 24S-hydroxycholesterol may explain the surprisingly high levels of this oxysterol in the circulation and is of interest in relation to the suggested role of 24S-hydroxycholesterol as a regulator of cholesterol homeostasis.

In agreement with previous work, treatment of rats with cholesterol, 2% in diet, stimulated the cholesterol 7 alpha-hydroxylase activity more than 2-fold. With less than 1% in diet, no significant effect was obtained. Intravenous infusion of cholesterol-enriched Intralipid had no stimulatory effect. In accordance with some recent work by other groups, it was shown that the stimulation of the cholesterol 7 alpha-hydroxylase by dietary cholesterol was associated with elevated levels of mRNA corresponding to the enzyme. Most of the stimulation of the activity induced by dietary cholesterol could not be prevented by lymphatic drainage. Feeding lymph fistulated rats with 2% cholesterol in diet stimulated the cholesterol 7 alpha-hydroxylase almost 2-fold, indicating that under the conditions employed, a major part of the cholesterol-induced stimulation of the activity was due to factor(s) unrelated to the flux of cholesterol from the intestine to the liver. There was a good correlation between the amount of cholesterol excreted in faeces and the activity of the cholesterol 7 alpha-hydroxylase. The half-life of intraperitoneally administered labelled cholic acid was significantly shorter in rats treated with 2% cholesterol in diet (t1/2 = 1.2 +/- 0.1 days) than in control rats (t1/2 = 1.9 +/- 0.18 days). A notable finding was that the weight of faeces was considerably higher in rats fed cholesterol than in the controls. It is hypothesized that a high dietary load of cholesterol causes increased binding of bile acids in the intestine and increased loss of bile acids in faeces. This leads to a reduced suppression of the cholesterol 7 alpha-hydroxylase by the bile acids. The results support the contention that the flux of bile acids rather than the flux of cholesterol from the intestine is the major direct regulator of bile acid biosynthesis.

Lymphatic drainage leads to a significant stimulation of both the cholesterol 7 alpha-hydroxylase and HMG-CoA reductase activity in rats (Björkhem et al. 1978. Biochem. Biophys. Res. Commun. 85: (532-540). This finding was confirmed here and it was also shown that ligation of the lymph duct leads to a similar but less pronounced effect. Ligation of the lymph duct or lymph fistulation of bile duct-ligated or cholestyramine-treated rats did not further increase 7 alpha-hydroxylase or the HMG-CoA reductase activity. However, treatment of lymph fistula rats with cholestyramine led to a significant further stimulation of both 7 alpha-hydroxylase and HMG-CoA reductase activity. Intravenous infusion of lymph into bile fistula rats led to a significant inhibition of both cholesterol 7 alpha-hydroxylase activity and HMG-CoA reductase activity. A corresponding infusion of cholesterol-enriched Intralipid led to inhibition of HMG-CoA reductase without effect on cholesterol 7 alpha-hydroxylase activity. The results show that cholesterol 7 alpha-hydroxylase is feedback-regulated by bile acids in a situation where the flux of cholesterol to the liver is interrupted also. The possibility is discussed that there is a factor in the lymph that down-regulates cholesterol 7 alpha-hydroxylase. If such a factor exists, it requires an intact enterohepatic circulation for its effect. The stimulatory effect of cholestyramine on HMG-CoA reductase also in lymph fistula rats shows that the previously demonstrated suppressive effect of bile acids on HMG-CoA reductase is not only due to the effect of bile acids on intestinal absorption of cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)