Bile acids are the end products of cholesterol metabolism secreted into bile. They are essential for the absorption of lipids and lipid soluble compounds from the intestine. Here we have identified a series of unusual Δ5-unsaturated bile acids in plasma and urine of patients with Smith-Lemli-Opitz syndrome (SLOS), a defect in cholesterol biosynthesis resulting in elevated levels of 7-dehydrocholesterol (7-DHC), an immediate precursor of cholesterol. Using liquid chromatography - mass spectrometry (LC-MS) we have uncovered a pathway of bile acid biosynthesis in SLOS avoiding cholesterol starting with 7-DHC and proceeding through 7-oxo and 7β-hydroxy intermediates. This pathway also occurs to a minor extent in healthy humans, but elevated levels of pathway intermediates could be responsible for some of the features SLOS. The pathway is also active in SLOS affected pregnancies as revealed by analysis of amniotic fluid. Importantly, intermediates in the pathway, 25-hydroxy-7-oxocholesterol, (25R)26-hydroxy-7-oxocholesterol, 3β-hydroxy-7-oxocholest-5-en-(25R)26-oic acid and the analogous 7β-hydroxysterols are modulators of the activity of Smoothened (Smo), an oncoprotein that mediates Hedgehog (Hh) signalling across membranes during embryogenesis and in the regeneration of postembryonic tissue. Computational docking of the 7-oxo and 7β-hydroxy compounds to the extracellular cysteine rich domain of Smo reveals that they bind in the same groove as both 20S-hydroxycholesterol and cholesterol, known activators of the Hh pathway.

Bile acids are the end products of cholesterol metabolism secreted into bile. They are essential for the absorption of lipids and lipid soluble compounds from the intestine. Here we have investigated the bile acid content of plasma and urine from patients with a defect in cholesterol biosynthesis, i.e. Smith-Lemli-Opitz syndrome (SLOS), resulting in elevated levels of 7-dehydrocholesterol (7-DHC), an immediate precursor of cholesterol. Using liquid chromatography – mass spectrometry (LC-MS) we have identified a novel pathway of bile acid biosynthesis in SLOS avoiding cholesterol starting with 7-DHC. This pathway also proceeds to a minor extent in healthy individuals. Monitoring of the pathway products could provide a rapid diagnostic for SLOS while elevated levels of pathway intermediates could be responsible for some of the features of the disease. Importantly, intermediates in the pathway are modulators of the activity of Smoothened, an oncoprotein that mediates Hedgehog signalling during embryogenesis and regeneration of postembryonic tissue.

Cholestenoic acids are formed as intermediates in metabolism of cholesterol to bile acids, and the biosynthetic enzymes that generate cholestenoic acids are expressed in the mammalian CNS. Here, we evaluated the cholestenoic acid profile of mammalian cerebrospinal fluid (CSF) and determined that specific cholestenoic acids activate the liver X receptors (LXRs), enhance islet-1 expression in zebrafish, and increase the number of oculomotor neurons in the developing mouse in vitro and in vivo. While 3β,7α-dihydroxycholest-5-en-26-oic acid (3β,7α-diHCA) promoted motor neuron survival in an LXR-dependent manner, 3β-hydroxy-7-oxocholest-5-en-26-oic acid (3βH,7O-CA) promoted maturation of precursors into islet-1+ cells. Unlike 3β,7α-diHCA and 3βH,7O-CA, 3β-hydroxycholest-5-en-26-oic acid (3β-HCA) caused motor neuron cell loss in mice. Mutations in CYP7B1 or CYP27A1, which encode enzymes involved in cholestenoic acid metabolism, result in different neurological diseases, hereditary spastic paresis type 5 (SPG5) and cerebrotendinous xanthomatosis (CTX), respectively. SPG5 is characterized by spastic paresis, and similar symptoms may occur in CTX. Analysis of CSF and plasma from patients with SPG5 revealed an excess of the toxic LXR ligand, 3β-HCA, while patients with CTX and SPG5 exhibited low levels of the survival-promoting LXR ligand 3β,7α-diHCA. Moreover, 3β,7α-diHCA prevented the loss of motor neurons induced by 3β-HCA in the developing mouse midbrain in vivo.Our results indicate that specific cholestenoic acids selectively work on motor neurons, via LXR, to regulate the balance between survival and death.

Cholic acid and 24( S ),25-epoxycholesterol are endogenous midbrain LXR ligands that are neurogenic for red nucleus or dopaminergic neurons, respectively.

Liver X receptors (LXRs) α and β are nuclear receptors whose transcriptional activity is regulated by oxysterols, the oxidized forms of cholesterol. Described in the late 1990s as lipid sensors, both LXRs regulate cholesterol and fatty acid homeostasis. Over the years, deep phenotypic analyses of mouse models deficient for LXRα and/or LXRβ have pointed out various other physiological functions including glucose homeostasis, immunology, and neuroprotection. This review enlightens the “endocrine” functions of LXRs; they deeply impact plasma glucose directly and by modulating insulin signaling, renin–angiotensin–aldosterone axis, thyroid and pituitary hormone levels, and bone homeostasis. Besides, LXR signaling is also involved in adrenal physiology, steroid synthesis, and male and female reproduction. Hence, LXRs are definitely involved in the endocrine system and could thus be considered as endocrine receptors, even though oxysterols do not fully correspond to the definition of hormones. Finally, because they are ligand-regulated transcription factors, LXRs are potential pharmacological targets with promising beneficial metabolic effects.

Bile acids constitute a large family of steroids in vertebrates, normally formed from cholesterol and carrying a carboxyl group in a side-chain of variable length. Bile alcohols, also formed from cholesterol, have similar structures as bile acids, except for the absence of a carboxyl group in the steroid skeleton. The conversion of cholesterol to bile acids and/or bile alcohols is of major importance for maintenance of cholesterol homeostasis, both from quantitative and regulatory points of view (Chiang, 2004; Kalaany and Mangelsdorf, 2006; Moore, Kato, Xie, et al., 2006; Scotti, Gilardi, Godio, et al., 2007). Appropriately conjugated bile acids and bile alcohols (also referred to as bile salts) are secreted in bile and serve vital functions in the absorption of lipids and lipid-soluble compounds (Hofmann, 2007). Reliable analytical methods are required for studies of the functions and pathophysiological importance of the variety of bile acids and bile alcohols present in living organisms. When combined with genetic and proteomic studies, analysis of these small molecules (in today’s terminology: metabolomics, steroidomics, sterolomics, cholanoidomics, etc.) will lead to a deeper understanding of the integrated metabolic processes in lipid metabolism.

The formation of bile acids/bile alcohols is of major importance for the maintenance of cholesterol homeostasis. Besides their functions in lipid absorption, bile acids/bile alcohols are regulatory molecules for a number of metabolic processes. Their effects are structure-dependent, and numerous metabolic conversions result in a complex mixture of biologically active and inactive forms. Advanced methods are required to characterize and quantify individual bile acids in these mixtures. A combination of such analyses with analyses of the proteome will be required for a better understanding of mechanisms of action and nature of endogenous ligands. Mass spectrometry is the basic detection technique for effluents from chromatographic columns. Capillary liquid chromatography-mass spectrometry with electrospray ionization provides the highest sensitivity in metabolome analysis. Classical gas chromatography-mass spectrometry is less sensitive but offers extensive structure-dependent fragmentation increasing the specificity in analyses of isobaric isomers of unconjugated bile acids. Depending on the nature of the bile acid/bile alcohol mixture and the range of concentration of individuals, different sample preparation sequences, from simple extractions to group separations and derivatizations, are applicable. We review the methods currently available for the analysis of bile acids in biological fluids and tissues, with emphasis on the combination of liquid and gas phase chromatography with mass spectrometry.

Cholesterol is the precursor of many compounds with functions in the physiology and metabolism of the organism. Methods for the multicomponent analysis of these compounds and their metabolites (metabolomics) are needed to improve our understanding of their roles in different species, organs, cells and metabolic situations and to clarify structure/activity relationships. This review discusses methods based on combinations of ion exchange and reversed-phase separations for sample preparation with derivatization and "charge-tagging" for chromatography-mass spectrometry in qualitative and quantitative characterizations of oxysterol, bile alcohol, bile acid, and steroid hormone metabolomes. Advantages, disadvantages and potential improvements for high-throughput applications are briefly discussed.

In this study we have profiled the free sterol content of cerebrospinal fluid by a combination of charge tagging and liquid chromatography-tandem mass spectrometry. Surprisingly, the most abundant cholesterol metabolites were found to be C27 and C24 intermediates of the bile acid biosynthetic pathways with structures corresponding to 7α-hydroxy-3-oxocholest-4-en-26-oic acid (7.170 ± 2.826 ng/ml, mean ± S.D., six subjects), 3β-hydroxycholest-5-en-26-oic acid (0.416 ± 0.193 ng/ml), 7α,x-dihydroxy-3-oxocholest-4-en-26-oic acid (1.330 ± 0.543 ng/ml), and 7α-hydroxy-3-oxochol-4-en-24-oic acid (0.172 ± 0.085 ng/ml), and the C26 sterol 7α-hydroxy-26-norcholest-4-ene-3,x-dione (0.204 ± 0.083 ng/ml), where x is an oxygen atom either on the CD rings or more likely on the C-17 side chain. The ability of intermediates of the bile acid biosynthetic pathways to activate the liver X receptors (LXRs) and the farnesoid X receptor was also evaluated. The acidic cholesterol metabolites 3β-hydroxycholest-5-en-26-oic acid and 3β,7α-dihydroxycholest-5-en-26-oic acid were found to activate LXR in a luciferase assay, but the major metabolite identified in this study, i.e. 7α-hydroxy-3-oxocholest-4-en-26-oic acid, was not an LXR ligand. 7α-Hydroxy-3-oxocholest-4-en-26-oic acid is formed from 3β,7α-dihydroxycholest-5-en-26-oic acid in a reaction catalyzed by 3β-hydroxy-Δ5-C27-steroid dehydrogenase (HSD3B7), which may thus represent a deactivation pathway of LXR ligands in brain. Significantly, LXR activation has been found to reduce the symptoms of Alzheimer disease (Fan, J., Donkin, J., and Wellington C. (2009) Biofactors 35, 239–248); thus, cholesterol metabolites may play an important role in the etiology of Alzheimer disease.

Pregnenolone (PREG) and dehydroepiandrosterone (DHEA), and their respective sulfated forms PREGS and DHEAS, were among the first steroids to be identified in rodent brain. However, unreliable steroid isolation and solvolysis procedures resulted in errors, particularly in the case of brain steroid sulfates analyzed by radioimmunology or GC-MS of liberated free steroids. By using a solid-phase extraction recycling/elution procedure, allowing the strict separation of sulfated, free, and fatty acid esters of PREG and DHEA, PREGS and DHEAS, unlike free PREG, were not detected in rat and mouse brain and plasma. Conversely, considerable amounts of PREG and DHEA were released from unknown precursor(s) present in the lipoidal fraction, distinct from fatty acid ester conjugates. Chromatographic and mass spectrometric studies of the nature of the precursor(s) showed that autoxidation of brain cholesterol (CHOL) was responsible for the release of PREG and DHEA from the lipoidal fraction. When inappropriate protocols were used, CHOL was also the precursor of PREG and DHEA obtained from the fraction assumed to contain sulfated steroids. In contrast, free PREG was definitely confirmed as an endogenous steroid in rat brain. Our study shows that an early removal of CHOL from brain extracts coupled to well-validated extraction and fractionation procedures are prerequisites for reliable measurements of free and conjugated PREG and DHEA by GC-MS or other indirect methods.

In this study two regions of embryonic (E11) mouse central nervous system (CNS) have been profiled for their unesterified sterol content. Using high-performance liquid chromatography (HPLC)-mass spectrometry (MS) and tandem mass spectrometry (MS(n)) low levels of oxysterols (estimated 2-165 ng g(-1) wet weight) were identified in cortex (Ctx) and spinal cord (Sc). The identified oxysterols include 7 alpha-, 7 beta-, 22R-, 24S-, 25- and 27-hydroxycholesterol; 24,25- and 24,27-dihydroxycholesterol; and 24S,25-epoxycholesterol. Of these, 24S-hydroxycholesterol is biosynthesised exclusively in brain. In comparison to adult mouse where the 24S-hydroxycholesterol level is about 40 microg g(-1) in brain the level of 24S-hydroxycholesterol reported here (estimated 26 ng g(-1) in Ctx and 13 ng g(-1) in Sc) is extremely low. Interestingly, the level of 24S,25-epoxycholesterol in both CNS regions (estimated 165 ng g(-1) in Ctx and 91 ng g(-1) in Sc) is somewhat higher than the levels of the hydroxycholesterols. This oxysterol is formed in parallel to cholesterol via a shunt of the mevalonate pathway and its comparatively high abundance may be a reflection of a high rate of cholesterol synthesis at this stage of development. Levels of cholesterol (estimated 1.25 mg g(-1) in Ctx and 1.15 mg g(-1) in Sc) and its precursors were determined by gas chromatography-mass spectrometry (GC-MS). In both CNS regions cholesterol levels were found to be lower than those reported in the adult, but in relation to cholesterol the levels of cholesterol precursors were higher than found in adult indicating a high rate of cholesterol synthesis. In summary, our data provide evidence for the presence of endogenous oxysterols in two brain regions of the developing CNS. Moreover, while most of the enzymes involved in hydroxysterol synthesis are minimally active at E11, our results suggest that the mevalonate pathway is significantly active, opening up the possibility for a function of 24S,25-epoxycholesterol during brain development.

In humans, the brain represents only about 2% of the body’s mass but contains about one-quarter of the body’s free cholesterol. Cholesterol is synthesized de novo in brain and removed by metabolism to oxysterols. 24S-Hydoxycholesterol represents the major metabolic product of cholesterol in brain, being formed via the cytochrome P450 (CYP) enzyme CYP46A1. CYP46A1 is expressed exclusively in brain, normally by neurons. In this study, we investigated the effect of 24S-hydroxycholesterol on the proteome of rat cortical neurons. With the use of two-dimensional liquid chromatography linked to nanoelectrospray tandem mass spectrometry, over 1040 proteins were identified including members of the cholesterol, isoprenoid and fatty acid synthesis pathways. With the use of stable isotope labeling technology, the protein expression patterns of enzymes in these pathways were investigated. 24S-Hydroxycholesterol was found to down-regulate the expression of members of the cholesterol/isoprenoid synthesis pathways including 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (EC 2.3.3.10), diphosphomevalonate decarboxylase (EC 4.1.1.33), isopentenyl-diphosphate delta isomerase (EC 5.3.3.2), farnesyl-diphosphate synthase (Geranyl trans transferase, EC 2.5.1.10), and dedicated sterol synthesis enzymes, farnesyl-diphosphate farnesyltransferase 1 (squalene synthase, EC 2.5.1.21) and methylsterol monooxygenase (EC 1.14.13.72). The expression of many enzymes in the cholesterol/isoprenoid and fatty acid synthesis pathways are regulated by the membrane-bound transcription factors named sterol regulatory element-binding proteins (SREBPs), which themselves are both transcriptionally and post-transcriptionally regulated. The current proteomic data indicates that 24S-hydroxycholesterol down-regulates cholesterol synthesis in neurons, possibly, in a post-transcriptional manner through SREBP-2. In contrast to cholesterol metabolism, enzymes responsible for the synthesis of fatty acids were not found to be down-regulated in neurons treated with 24S-hydroxycholesterol, while apolipoprotein E (apo E), a cholesterol trafficking protein, was found to be up-regulated. Taken together, this data leads to the hypothesis that, in times of cholesterol excess, 24S-hydroxycholesterols signals down-regulation of cholesterol synthesis enzymes through SREBP-2, but up-regulates apo E synthesis (through the liver X receptor) leading to cholesterol storage and restoration of cholesterol balance.

Mammalian antimicrobial peptides contribute to the protective barrier against microbes at epithelial surfaces. This study focuses on the promoter of the human CAMP gene, encoding the antimicrobial peptide LL-37, and induction of the gene in the colonic epithelial cell line HT-29. CAMP promoter segments were inserted in front of a luciferase reporter in order to identify regulatory regions. A transcription promoting region was identified and the transcription factor PU.1 of the Ets family was recruited to this region as shown by ChIP analysis. This ties PU.1 to the regulation of human innate epithelial defences for the first time. In addition, the conserved second intron was found to exert a transcription enhancing effect in cooperation with the 3′ end of the proximal promoter, and the importance of two upstream AUG codons was examined. Moreover, we here demonstrate that lithocholic acid enhances CAMP transcription, and does so additively with butyrate. Thus, a crosstalk between bacteria and host epithelia of the gut could be partially mediated via these two bacterial products to obtain gut homeostasis.

While the proteome defines the expressed gene products, the metabolome results from reactions controlled by such gene products. Plasma represents an accessible "window" to the metabolome both in regard of availability and content. The wide range of the plasma metabolome, in terms of molecular diversity and abundance, makes its comprehensive analysis challenging. Here we demonstrate an analytical method designed to target one region of the metabolome, that is, oxysterols. Since the discovery of their biological activity as ligands to nuclear receptors there has been a reawakening of interest in oxysterols and their analysis. In addition, the oxysterols, 24S- and 27-hydroxycholesterol, are currently under investigation as potential biomarkers associated with neurodegenerative disorders such as Alzheimer's disease and multiple sclerosis; widespread analysis of these lipids in clinical studies will require the development of robust, sensitive and rapid analytical techniques. In this communication we present results of an investigation of the oxysterols content of human plasma using a newly developed high-performance liquid chromatography-mass spectrometry (HPLC-MS) method incorporating charge-tagging and high-resolution MS. The method has allowed the identification in plasma of monohydroxylated cholesterol molecules, 7 alpha-, 24S-, and 27-hydroxycholesterol; the cholestenetriol 7 alpha,27-dihydroxycholesterol; and 3 beta-hydroxycholest-5-en-27-oic acid and its metabolite 3 beta,7 alpha-dihydroxycholest-5-en-27-oic acid. The methodology described is also applicable for the analysis of other sterols in plasma, that is, cholesterol, 7-dehydrocholesterol, and desmosterol, as well as cholesterol 5,6-seco-sterols and steroid hormones. Although involving derivatization, sample preparation is straightforward and chromatographic analysis rapid (17 min), while the MS method offers high sensitivity (ng/mL of sterol in plasma, or pg on-column) and specificity. The methodology is suitable for targeted metabolomic analysis of sterols, oxysterols, and steroid hormones opening a "window" to view this region of the metabolome.

Pregnenolone sulfate (PREGS) has been shown, either at high nanomolar or at micromolar concentrations, to increase neuronal activity by inhibiting GABAergic and by stimulating glutamatergic neurotransmission. PREGS is also a potent modulator of sigma type 1 (sigma1) receptors. It has been proposed that these actions of PREGS underlie its neuropharmacological effects, and in particular its influence on memory processes. On the other hand, the PREGS-mediated increase in neuronal excitability may become dangerous under particular conditions, for example in the case of excitotoxic stress or convulsions. However, the physiopathological significance of these observations has recently been put into question by the failure to detect significant levels of PREGS within the brain and plasma of rats and mice, either by direct analytical methods based on liquid chromatography/mass spectrometry (LC/MS) or enzyme linked immunosorbent assay (ELISA) with specific antibodies against PREGS, or by indirect gas chromatography/mass spectrometry (GC/MS) analysis with improved sample workup. These recent results have not come to the attention of a large number of neurobiologists interested in steroid sulfates. However, although available direct analytical methods have failed to detect levels of PREGS above 0.1-0.3 ng/g in brain tissue, it may be premature to completely exclude the local formation of biologically active PREGS within specific and limited compartments of the nervous system. In contrast to the situation in rodents, significant levels of sulfated 3beta-hydroxysteroids have been measured in human plasma and brain. Previous indirect measures of steroid sulfates by radioimmunoassays (RIA) or GC/MS had detected elevated levels of PREGS in rodent brain. The discrepancies between the results of different assay procedures have revealed the danger of indirect analysis of steroid sulfates. Indeed, PREGS must be solvolyzed/hydrolyzed prior to RIA or GC/MS analysis, and it is the released, unconjugated PREG which is then quantified. Extreme caution needs to be exercised during the preparation of samples for RIA or GC/MS analysis, because the fraction presumed to contain only steroid sulfates can be contaminated by nonpolar components from which PREG is generated by the solvolysis/hydrolysis/derivatization reactions.

Fischler B, Bodin K, Stjernman H, Olin M, Hansson M, Sjovall J, Bjorkhem T (Karolinska University Hospital, Huddinge; Karolinska Institutet, Stockholm; and County Hospital Ryhov, Jonkoping; Sweden). Cholestatic liver disease in adults may be due to an inherited defect in bile acid biosynthesis. J Intern Med 2007; 262: 254-262.Background. An increasing number of treatable inborn errors of bile acid synthesis have been described, primarily in infants with severe cholestatic liver disease.Results. The present patient, whose two older siblings had died from progressive cholestatic liver disease, developed neonatal cholestasis and rickets but recovered during the childhood years and follow-up was terminated at 12 years of age. The patient presented again at 26 years of age with jaundice and pathological liver function tests. This was normalized upon treatment with ursodeoxycholic acid. Electrospray mass spectrometry of urine showed predominance of unsaturated bile acids, characteristic of 3 beta-hydroxy-Delta(5)-C-27-steroid dehydrogenase/isomerase (HSD3B7) deficiency. The activity of HSD3B7 in cultured fibroblasts was less than 5% of normal. A single homozygous mutation was found in exon 4 leading to an amino acid exchange (S162R) and loss of enzyme activity.Conclusion. This case illustrates that infants with an inherited absence of HSD3B7 may survive the neonatal period of life and childhood without treatment with bile acids. A low level of sulphation of the abnormal trihydroxy bile acid formed as a result of enzyme deficiency may be of importance for survival. The possibility that liver disease presenting in the adult may be due to a mutation in the HSD3B7 gene should be considered, especially in cases with familial occurrence of liver disease and earlier periods of liver dysfunction.