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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.

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.

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.

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 humans, the brain accounts for about 20% of the body's free cholesterol, most of which is synthesized de novo in brain. To maintain cholesterol balance throughout life, cholesterol becomes metabolized to 24S-hydroxycholesterol, principally in neurons. In mouse, rat, and probably human, metabolism to 24S-hydroxycholesterol accounts for about 50% of cholesterol turnover; however, the route by which the remainder is turned over has yet to be elucidated. Here, we describe a novel liquid chromatography (LC) multi-stage fragmentation mass spectrometry (MS(n)) methodology for the identification, with high sensitivity (low pg), of cholesterol metabolites in rat brain. The methodology includes derivatization to enhance ionization, exact mass analysis at high resolution to identify potential metabolites, and LC-MS(n) (n=3) to allow their characterization. 24S-hydroxycholesterol was confirmed as a major oxysterol in rat brain, and other oxysterols identified for the first time in brain included 24,25-, 24,27-, 25,27-, 6,24,- 7alpha,25-, and 7alpha,27-dihydroxycholesterols. In addition, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al and its aldol, two molecules linked to amyloidogenesis of proteins, were characterized in rat brain.

Oxysterols are oxygenated derivatives of cholesterol. They are intermediates in cholesterol excretion pathways and may also be regarded as transport forms of cholesterol. The introduction of additional hydroxyl groups to the cholesterol skeleton facilitates the flux of oxysterols across the blood brain barrier, and oxysterols have been implicated in mediating a number of cholesterol-induced metabolic effects. Oxysterols are difficult to analyze by atmospheric pressure ionization mass spectrometry on account of the absence of basic or acidic functional groups in their structures. In this communication, we report a method for the derivatization and analysis of oxysterols by electrospray mass spectrometry. Oxysterols with a 3beta-hydroxy-Delta5 structure were converted by cholesterol oxidase to 3-oxo-Delta4 steroids and then derivatized with the Girard P reagent to give Girard P hydrazones, which were subsequently analyzed by tandem mass spectrometry. The improvement in sensitivity for the analysis of 25-hydroxycholesterol upon oxidation and derivatization was over 1000.

Farnesoid X receptor knockout (Fxr(-/-)) mice cannot upregulate the bile salt export pump in bile acid loading or cholestatic conditions. To investigate whether Fxr(-/-) mice differ in bile acid detoxification compared with wild-type mice, we performed a comprehensive analysis of bile acids extracted from liver, bile, serum, and urine of naive and common bile duct-ligated wild-type and Fxr(-/-) mice using electrospray and gas chromatography mass spectrometry. In addition, hepatic and renal gene expression levels of Cyp2b10 and Cyp3a11, and protein expression levels of putative renal bile acid-transporting proteins, were investigated. We found significantly enhanced hepatic bile acid hydroxylation in Fxr(-/-) mice, in particular hydroxylations of cholic acid in the 1beta, 2beta, 4beta, 6alpha, 6beta, 22, or 23 position and a significantly enhanced excretion of these metabolites in urine. The gene expression level of Cyp3a11 was increased in the liver of Fxr(-/-) mice, whereas the protein expression levels of multidrug resistance-related protein 4 (Mrp4) were increased in kidneys of both genotypes during common bile duct ligation. In conclusion, Fxr(-/-) mice detoxify accumulating bile acids in the liver by enhanced hydroxylation reactions probably catalyzed by Cyp3a11. The metabolites formed were excreted into urine, most likely with the participation of Mrp4.

Neutral steroids are difficult to analyse using desorption ionisation methods coupled with mass spectrometry (MS). However, steroids with an unhindered ketone group can readily be derivatised with the Girard P (GP) reagent to give GP hydrazones. Steroid GP hydrazones contain a quaternary nitrogen atom and are readily desorbed in the matrix-assisted laser desorption/ionisation (MALDI) process, giving an improvement in sensitivity of two orders of magnitude. Steroids without a ketone group, but with a 3beta-hydroxy-Delta5 function, can be readily converted to 3-oxo-Delta4 steroids and subsequently derivatised to GP hydrazones for MALDI analysis. In addition to giving strong [M]+ ions upon MALDI, steroid GP hydrazones give informative post-source decay (PSD) spectra. By using the accurate mass of the precursor-ion measured by MALDI-MS, in combination with the structural information encoded in its PSD spectrum, steroid structures can readily be determined.

A new sample preparation method coupled to GC-MS analysis was developed and validated for quantification of sulfate esters of pregnenolone (PREG-S) and dehydroepiandrosterone (DHEA-S) in rat brain. Using a solid-phase extraction recycling protocol, the results show that little or no PREG-S and DHEA-S (

Cholesterol and its metabolites, e.g., steroid hormones and bile acids, constitute a class of compounds of great biological importance. Their chemistry, biochemistry, and regulation in the body have been intensely studied for more than two centuries. The author has studied aspects of the biochemistry and clinical chemistry of steroids and bile acids for more than 50 years, and this paper, which is an extended version of the Schroepfer Medal Award lecture, reviews and discusses part of this work. Development and application of analytical methods based on chromatography and mass spectrometry (MS) have been a central part of many projects, aiming at detailed characterization and quantification of metabolic profiles of steroids and bile acids under different conditions. In present terminology, much of the work may be termed steroidomics and cholanoidomics. Topics discussed are bile acids in human bile and feces, bile acid production, bacterial dehydroxylation of bile acids and steroids during the enterohepatic circulation, profiles of steroid sulfates in plasma of humans and other primates, development of neutral and ion-exchanging lipophilic derivatives of Sephadex for sample preparation and group separation of steroid and bile acid conjugates, profiles of steroids and bile acids in human urine under different conditions, hydroxylation of bile acids in liver disease, effects of alcohol-induced redox changes on steroid synthesis and metabolism, alcohol-induced changes of bile acid biosynthesis, compartmentation of bile acid synthesis studied with 3H-labeled ethanol, formation and metabolism of sulfated metabolites of progesterone in human pregnancy, abnormal patterns of these in patients with intrahepatic cholestasis of pregnancy corrected by ursodeoxycholic acid, inherited and acquired defects of bile acid biosynthesis and their treatment, conjugation of bile acids and steroids with N-acetylglucosamine, sulfate-glucuronide double conjugates of hydroxycholesterols, extrahepatic 7alpha-hydroxylation and 3-dehydrogenation of hydroxycholesterols, and extrahepatic formation of C27 bile acids. The final part discusses analysis of free and sulfated steroids in brain tissue by capillary liquid chromatography-electrospray MS and suggests a need for reevaluation of the function of steroid sulfates in rat brain.

A method designed for the analysis of sulfated neurosteroids and unconjugated ketonic neurosteroids in rat brain using nanoscale liquid chromatography-electrospray (nano-LC-ES) mass spectrometry is described. Neurosteroids in rat brain tissue were extracted, purified, and separated into two groups, neutral unconjugated steroids and steroid sulfates, by employing solid-phase partition, cation- and anion-exchange chromatography. The steroid sulfate fraction was analyzed by nano-LC-ES mass spectrometry. Contrary to expectations, the sulfates of pregnenolone and dehydroepiandrosterone (DHEA) were not detected. Internal standards, including pregnenolone sulfate, were recovered and the detection limit of the method was 0.3 ng/g of wet brain. Cholesterol sulfate was detected at a level of 1.2 microg/g of wet brain. The neutral unconjugated steroid fraction was derivatized with hydroxylamine hydrochloride to convert oxosteroids into their oximes. The oximes were isolated using cation-exchange chromatography and were analyzed by nano-LC-ES tandem mass spectrometry. The analyses of the neutral unconjugated steroid fraction confirmed the presence in rat brain of pregnenolone, pregnanolone isomers, progesterone, testosterone, and DHEA, which were characterized by their retention times, the mass of the protonated molecules, and characteristic fragment ions. The levels were estimated by addition of [3,4-(13)C(2)]-progesterone as an internal standard and found to be in a range of 0.04-20 ng/g.

The identification, quantification and localisation of steroids in biological fluids and tissues are subjects of considerable importance. Not only do steroids have classical hormonal properties via binding to nuclear receptors, they can also elicit cellular responses via interactions with other proteins. For mass spectrometric analysis, neutral steroids are not readily ionised by either electrospray (ES) or matrix-assisted laser desorption/ionisation (MALDI). In this communication a derivatisation protocol is presented which allows for the rapid analysis of neutral oxosteroids by both ES and MALDI mass spectrometry. Neutral oxosteroids are derivatised to Girard P hydrazones. When analysed by tandem mass spectrometry the derivatised steroids fragment to give structurally informative spectra allowing subsequent steroid identification. The derivatisation method is simple, the reagents are commercially available, and reaction products are easily isolated from the reaction mixture. Analyte identification can be performed at the sub-pg level.

Background In this open, randomized study, the pharmacokinetics, clinical efficacy, and safety of a 48-h continuous interscalene infusion of 2 mg/ml ropivacaine for postoperative pain relief were investigated in patients undergoing open major shoulder surgery. Methods An initial interscalene block with 30 ml ropivacaine, 7.5 mg/ml (225 mg), was performed. After completion of interscalene block, all patients (n = 24) received general anesthesia, and 6 h after interscalene block, a 48-h continuous interscalene infusion of 12 or 18 mg/h using 2 mg/ml ropivacaine was started. Total and unbound plasma concentrations of ropivacaine and 2.6-pipecoloxylidide (PPX; a major active metabolite) were determined during and up to 6 h after the interscalene infusion. Postoperative pain at rest was assessed by a visual analog scale. Supplementary analgesics and adverse events were recorded. Results Plasma concentrations of total and unbound ropivacaine were proportional to the total dose. At the end of the interscalene infusion of 9 ml/h, the mean +/- SD plasma concentrations of total and unbound ropivacaine were 1.40 +/- 0.54 and 0.03 +/- 0.01 mg/l, respectively, and of total and unbound PPX were 0.70 +/- 0.38 and 0.30 +/- 0.20 mg/l, respectively. Plasma concentrations of unbound ropivacaine and unbound PPX, added together, remained well below threshold levels for systemic central nervous system toxicity. There were no significant differences between the groups for postoperative pain (median maximum of about 20 mm on the visual analog scale in both groups), analgesic consumption, or quality of pain relief assessed by the patient. No signs or symptoms of systemic local anesthetic toxicity were observed. Conclusion A 48-h continuous interscalene infusion of 6 or 9 ml/h ropivacaine, 2 mg/ml, started 6 h after an initial interscalene block of 30 ml ropivacaine, 7.5 mg/ml, provided satisfactory postoperative pain relief after major shoulder surgery and was well tolerated. Unbound plasma concentrations of ropivacaine and PPX remained well below threshold levels for systemic central nervous toxicity.

Aims This study investigated the absorption characteristics and the tolerability of rectally administered ropivacaine, a local anaesthetic, intended as a new local therapy for ulcerative colitis. Methods Thirty-two healthy volunteers participated in a randomized, placebo-controlled study. In study phase 1 (n = 16, double-blind, crossover) single rectal doses of ropivacaine corresponding to 50, 100 and 200 mg base were given as 20-ml gel enemas. Eight of these subjects also received an i.v. infusion corresponding to 20 mg (2H3)ropivacaine base given with the last rectal dose. In study phase 2 (n = 16, single-blind, crossover) the same rectal doses were given but formulated in 20, 40 and 80 ml gel, respectively. Peripheral venous plasma samples and urine were collected over 12 h after dosing and analysed for ropivacaine base by gas chromatography and (2H3)ropivacaine by gas chromatography-mass spectrometry. Ropivacaine and metabolites were analysed in urine by reversed phase liquid chromatography. Results AUC was proportional to the dose with a point estimate [95% confidence interval (CI)] for the increase, after doubling the dose, of 1.91 (1.66–2.20) and 1.95 (1.78–2.13) in study phases 1 and 2, respectively. The increase in Cmax was also proportional to the dose with corresponding results of 1.76 (1.52–2.04) and 1.84 (1.70–1.99). The volume of the rectal formulation had no influence on either the extent or the time course of absorption. The mean (s.d.) absolute bioavailability (%F) was 56 (18)%. AUC and Cmax showed a two- to three-fold lower intra- than interindividual variability. Zero-order kinetics dominated the first 4 h of the absorption phase. Thereafter first-order kinetics were observed. The terminal half-lives were similar between the rectal doses and were longer than that after the i.v. administration, indicating an absorption-dependent half-life. The main urinary metabolite was 3-hydroxyropivacaine corresponding to about 23% of the dose. The subjects had no difficulties in retaining the doses and rectal administration of ropivacaine was well tolerated, both locally and systemically. Conclusions Plasma drug concentrations were proportional to the dose after rectally administered doses corresponding to 50–200 mg ropivacaine base in a gel formulation. The drug was well-tolerated. Mean bioavailability was about 60% and not influenced by variations in the enema volume. Initial absorption seemed to follow zero-order kinetics and then first-order kinetics after about 4 h. Cmax and AUC showed considerably less intra- compared with inter-individual variability, resulting in more consistent plasma concentrations within subjects.

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.

The retinoid X receptor (RXR) is a nuclear receptor that functions as a ligand-activated transcription factor. Little is known about the ligands that activate RXR in vivo. Here, we identified a factor in brain tissue from adult mice that activates RXR in cell-based assays. Purification and analysis of the factor by mass spectrometry revealed that it is docosahexaenoic acid (DHA), a long-chain polyunsaturated fatty acid that is highly enriched in the adult mammalian brain. Previous work has shown that DHA is essential for brain maturation, and deficiency of DHA in both rodents and humans leads to impaired spatial learning and other abnormalities. These data suggest that DHA may influence neural function through activation of an RXR signaling pathway.

A method for the analysis of neutral oxosteroids by electrospray mass spectrometry is described. The oxosteroids are converted into their oximes by treatment with hydroxyammonium chloride in aqueous methanol. Intense peaks corresponding to protonated oxime molecules are observed in nano-electrospray mass spectra. The detection limits for the oximes of progesterone, pregnenolone and dehydroepiandrosterone were 2.5, 5 and 25 pg/microL, respectively, approximately 20 times lower than for the underivatised steroids. The signal intensities were proportional to the concentration of the steroids in the range of 500 to 2.5 pg/microL. Fragmentation by collision-induced dissociation (CID) was studied using oximes of 28 model steroids carrying an oxo group at C-3, C-17 or C-20. Some of the steroid oximes were labelled with deuterium or (15)N. Fragment ions were observed which yielded useful structural information. Upon CID, protonated oximes of 3-oxo-Delta(4)-steroids produced abundant ions by cleavage through the B-ring and by loss of the side chain, while protonated oximes of saturated 3-oxosteroids did not give abundant ions by cleavage through the B-ring. Protonated oximes of 20-oxosteroids unsubstituted at C-21, C-17 or C-16 produced a characteristic ion at m/z 86 containing the side chain, C-16 and C-17. Protonated oximes of steroids containing only a 17-oxo group gave fewer ions of diagnostic value. Coupled with the selective isolation of steroid oximes from a biological matrix this method of derivatisation and CID may be used for the analysis of neutral oxosteroids in biological samples.

The pathogenesis of intrahepatic cholestasis of pregnancy (ICP) can be related to abnormalities in the metabolism and disposition of sex hormones and/or bile acids, determined by a genetic predisposition interacting with environmental factors. The total amount of oestrogens and progesterone circulating in the blood or excreted in the urine of ICP patients is similar to normal pregnancies. Thus, the search for the cause has been focused on abnormal hormone metabolites. The cholestatic potential of some D-ring oestrogen metabolites is supported by experimental and clinical data. Similar observations with regard to bile acids and progesterone metabolites are still scarce. This article reviews current knowledge in this field, including our own data. Bile acid synthesis appears to be reduced in patients with ICP, in whom primary conjugated bile acids are retained in blood. The major bile acid in blood and urine of these patients is cholic acid instead of chenodeoxycholic acid present in normal pregnancies. Hydroxylation and sulfation of bile acids are enhanced, while glucuronidation appears to be of lesser importance. The synthesis of progesterone appears unimpaired, while the profiles of progesterone metabolites in plasma and urine are different from normal pregnancies, with a larger proportion of mono- and disulfated metabolites, mainly 3alpha,5alpha isomers. Glucuronidated metabolites, however, are unchanged. With the administration of ursodeoxycholic acid (UDCA) to patients with ICP, pruritus and serum liver values are improved, the concentration of bile acids in blood is diminished and the proportion of their conjugated metabolites returned to normal. Simultaneously, the concentration of sulfated progesterone metabolites in blood and their urinary excretion are reduced. The serum levels of bile acids and progesterone metabolites before UDCA administration and their decrease during treatment do not correlate with each other. We propose that patients with ICP have a selective defect in the secretion of sulfated progesterone metabolites into bile and speculate that this may be caused by genetic polymorphism of canalicular transporter(s) for steroid sulfates or their regulation. Interaction with oestrogen metabolites and/or some exogenous compounds may further enhance the process triggering ICP in genetically predisposed individuals.

Progesterone inhibits intracellular transport of lysosomal cholesterol in cultured cells, and thus at least in part mimics the biochemical phenotype of Niemann-Pick type C disease (NPC) in human fibroblasts. The goal of this study was to determine whether metabolism of progesterone to other steroids is affected by the NPC mutation or by P-glycoprotein (a known progesterone target). We found that human fibroblasts metabolize progesterone in three steps: rapid conversion to 5alpha-pregnane-3,20-dione, which is then reduced to 5alpha-pregnane-3beta(alpha)-ol-20-one with subsequent 6alpha-hydroxylation. The pattern and rates of progesterone metabolism were not significantly different in a variety of fibroblasts from normal individuals, NPC patients, and obligate heterozygotes. Inhibition of steroid 5alpha-reductase with finasteride completely blocked metabolism of progesterone but had no effect on inhibition of LDL-stimulated cholesterol esterification (IC50 = 10 microM). Progesterone also partially inhibited 25-hydroxycholesterol-induced cholesterol esterification, with similar dose-dependence in normal and NPC fibroblasts. P-glycoprotein levels varied significantly among the various fibroblasts tested, but no correlation with NPC phenotype or rate of progesterone metabolism was noted, and P-glycoprotein inhibitors did not affect conversion of progesterone to products. These results indicate that metabolism of progesterone in human fibroblasts is largely independent of its ability to interfere with cholesterol traffic and P-glycoprotein function.

The value of derivatizing peptides at the C-terminus with 4-aminonaphthalenesulphonic acid (ansa) to aid in the de novo sequencing of peptides by mass spectrometry was assessed. Negative-ion electrospray of peptides is enhanced by derivatization witb ansa. The derivatizing group also has the effect of increasing the mass of the peptides by 205 Da, often shifting their deprotonated molecules to regions of the mass spectrum with reduced chemical noise. Collision-induced dissociation of naphthalenesulphonated peptide [M-H] - ions results in abundant fragment ions formed by charge-remote fragmentations. The resulting fragmentation patterns are less complex than those of the underivatized analogues and are easier to interpret. Peptides were successfully derivatized at the low picomole level and sequenced on the hundred femtomole level using nano-electrospray tandem mass spectrometry.

Nano-electrospray has been used in combination with high resolution and tandem mass spectrometry in the analysis of the major mercapturic acid pathway metabolites of benzo[a]pyrene (BP). Accurate mass measurements indicate that the [M-H]- ion of the major metabolite has a chemical formula C25H22NO6S, which corresponds to the deprotonated form of tetrahydro-trihydroxy-BP-S-N-acetylcysteine. Tandem mass spectrometry of this [M-H]- ion results in a collision induced dissociation spectrum identical to that of synthetic 7,8,9,10-tetrahydro-8,9,10-trihydroxy-BP-7-S-N-acetylcysteine.

The concentrations in serum of sulfated metabolites of progesterone are known to be elevated in patients with intrahepatic cholestasis of pregnancy (ICP). The profiles of these metabolites and conjugated bile acids were analyzed in serum from 11 patients with ICP before and during administration of ursodeoxycholic acid (UDCA) (8 patients) or placebo (3 patients). The clinical condition of 7 of the patients given UDCA improved markedly, and 1 patient given placebo had a spontaneous remission of the disease. The total concentration of conjugated bile acids in the 11 patients was 25 +/- 6 micromol/L (mean +/- SEM) and decreased to 6.3 +/- 3.5 micromol/L in the 7 patients responding to treatment with UDCA. The level of 7alpha-hydroxy-4-cholesten-3-one was significantly lower (7.2 +/- 2.2 ng/mL) in patients with ICP than in healthy pregnancy (18 +/- 4.6 ng/mL) (P < .05). The concentrations of 5alpha-pregnane-3alpha,20alpha-diol mono- and disulfates decreased by 52% +/- 7.9% and 68% +/- 5.5%, respectively, in the patients responding to treatment. Similar decreases were observed for the mono- and disulfates of 5alpha-pregnane-3alpha,20alpha,21-triol and 5beta-pregnane-3alpha,20alpha-diol. The disulfate of 5alpha-pregnane-3beta,20alpha-diol showed a smaller decrease, while glucuronidated steroids were not affected. The 3alpha-/3beta-hydroxysteroid ratio and di-/monosulfate ratio decreased significantly during UDCA. The magnitudes of the changes of bile acid and steroid concentrations during UDCA were not correlated to each other. The results suggest that UDCA stimulates the biliary excretion of steroids with a 3alpha-sulfoxy group and disulfates. This effect seems to be independent of the effect on bile acid excretion, indicating the use of different transport proteins. The possibility of an effect of UDCA on the formation of the steroid sulfates cannot be excluded.

Background/Aims and Methods: The etiology of intrahepatic cholestasis of pregnancy (ICP) is unknown. We have performed comprehensive chromatographic and mass spectrometric analyses of progesterone metabolites and bile acids in serum and urine of six patients in order to characterize changes that might be of importance for the development of the disease. Results: Conjugated bile acids were increased in serum and urine of patients with ICP while the levels of unconjugated bile acids were similar in healthy pregnancies and ICP. Unconjugated and conjugated 7α,12α-dihydroxy-3-oxo-4-cholenoic acid was excreted in urine both in healthy pregnancies and in ICP, possibly indicating a rate limitation of 3-oxo-Δ 4 -steroid 5β-reductase in pregnancy. The serum levels and urinary excretion of total sulfated progesterone metabolites were increased in ICP while the glucuronides were unchanged or low. Confirming previous results, the fraction of metabolites with 3α-hydroxy-5α(H) configuration was increased. The urinary excretion of 5α-pregnane-3α,20α-diol 3-sulfate,20- N -acetylglucosaminide was greatly increased in ICP, as was that of 3α-hydroxy-5α-androstane-17β-carboxylic acid, assumed to be a progesterone metabolite. Conclusions: The combined results of this and previous studies are compatible with a primary change in the reductive metabolism of progesterone in ICP, resulting in increased formation of metabolites with a 3α-hydroxy-5α(H) configuration and a larger fraction of sulfates. There also seems to be a selective defect in the biliary secretion of sulfated metabolites, particularly disulfates.

Oxygenated derivatives of sterols (oxysterols), including 25-hydroxycholesterol and 27-hydroxycholesterol, have immunosuppressive effects. Oxysterols can directly induce apoptosis in immature thymocytes, cells which are inherently sensitive to induction of programmed cell death. For that reason, the metabolism of 25-hydroxycholesterol and 27-hydroxycholesterol in mouse thymus has been studied. When incubated with thymic tissue, both oxysterols were found to be 7alpha-hydroxylated with subsequent oxidation to 7alpha-hydroxy-3-oxo-delta4 steroids. A minor fraction of 27-hydroxycholesterol was also metabolised to 3beta-hydroxy-5-cholestenoic, 3beta,7alpha-dihydroxy-5-cholestenoic and 7alpha-hydroxy-3-oxo-4-cholestenoic acids. The 7alpha-hydroxylase was found to be localised to the thymic epithelial cells and the reaction was stimulated by interleukin-1beta and inhibited by metyrapone and RU486. In contrast to 25-hydroxycholesterol and 27-hydroxycholesterol, the 7alpha-hydroxylated metabolites, 7alpha,25-dihydroxycholesterol, 7alpha,25-dihydroxy-4-cholesten-3-one and 7alpha,27-dihydroxy-4-cholesten-3-one did not induce thymocyte apoptosis. The results suggest that 7alpha-hydroxylation may be of regulatory importance, possibly by protecting the developing thymocytes against toxic effects by oxysterols.

Solid-phase extraction and group separation by anion exchange chromatography were combined with capillary column liquid chromatography-mass spectrometry (LC/MS) to permit a thorough characterization of bile acids and intact conjugates of bile alcohols in human urine. Groups of compounds were separated according to acid strength and were analysed on a capillary column, 0.25 x 500 mm, packed with 5 microns particles of Chromasil C18, and connected via a fused silica capillary to the continuous-flow fast atom bombardment (CF-FAB) or electrospray (ES) sources of an AutoSpec-TOFFPD hybrid mass spectrometer. Acetonitrile:water mixtures containing 30 mM ammonium acetate pH 7.2 were used as mobile phases, with 5% glycerol added for FAB Ionisation. Bile acids were analysed directly or after derivatization of carboxyl groups with 4-aminobenzenesulphonic acid. Negative-ion spectra (m/z 1000 or 800 to 300 or 100) were recorded using the point detector or, in the case of ES ionization, the focal plane array detector (FPD). Deprotonated molecules of bile acids containing a sulphonic acid group were detected with a spectral signal to noise ratio of 5:1 when about 90 fmol were injected onto the column of the LC/CF-FAB system. The corresponding peak in the reconstructed ion chromatogram gave a signal-to-noise ratio of about 25:1. The sensitivity could be increased 20-50 times by using ES ionization and the FPD. Bile acids without a sulphonic acid group gave about 70% of the signal of sulphonic acids using ES ionization. The capillary column LC/MS systems were evaluated by analyses of urine from an infant with cholestatic liver disease. More than 150 different bile acids and bile alcohol conjugates were detected, some of which were partially characterized using collision induced dissociation (CID) of the deprotonated molecules and B/E linked scans. A number of compounds were detected for the first time, e.g. di-, tri-, and tetra-hydroxycholestanoic acids conjugated with N-acetylhexosamine and cholestenediol, cholestenetriol and cholestanetriol doubly conjugated with sulphuric acid and glucuronic acid. The relative merits of ES and FAB ionization are discussed.

Studies of bile acid transport systems require radio-labeled taurine-conjugated bile acids with high specific activity. An established synthetic procedure was optimized to provide mild, fast, and effective conjugation of radio-labeled taurine with different types of bile acids, including those with labile 7α-hydroxy-3-oxo-Δ4 or 3β,7α-dihydroxy-Δ5 structures. Taurine labeled with 14C or 3H was reacted with excess bile acid anhydride formed from the tributylamine salt and ethylchloroformate (2/1 M/M) in aqueous dioxane for 15 min at room temperature. The yields were higher than 95% and less than 2% side products were formed. Bile acid sulfates were conjugated with 14C- or 3H- labeled taurine by using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as the coupling reagent. The products were effectively purified by chromatography of the sodium salts on Sephadex LH-20. The yields of taurine-conjugated bile acid sulfates were 65–70%. © 1997 John Wiley & Sons, Ltd.

A series of pregnanediols and pregnanetriols doubly conjugated with N-acetylglucosamine and glucuronic or sulfuric acid has been identified in urine from pregnant women. Steroid conjugates were separated by ion-exchange chromatography and the glucuronide and monosulfate fractions were analysed by fast atom bombardment mass spectrometry. After removal of the acid moiety, the neutral steroids were isolated, derivatized, and analysed by gas chromatography-mass spectrometry (GC-MS). The analyses revealed the presence of steroids conjugated with N-acetylhexosamine both in the glucuronide and the monosulfate fractions. Following enzyme hydrolysis, the sugar was identified by GC-MS as N-acetylglucosamine (GlcNAc). The major steroid conjugated with GlcNAc both in the glucuronide and monosulfate fractions was identified as 5alpha-pregnane-3alpha,20alpha-diol. 5beta-Pregnane-3alpha,2Oalpha-diol was also present as a GlcNAc conjugate in both fractions whereas a GlcNAc conjugate of 5alpha-pregnane-3beta,20alpha-diol was only found in the sulfate fraction. 5alpha-Pregnane-3alpha,20alpha,21-triol was a double conjugate with GlcNAc in the sulfate fraction whereas a pregnane-2,3,20-triol was a double conjugate in the glucuronide fraction. The positions of conjugation were determined by collision-induced dissociation of the pseudomolecular anions produced by fast atom bombardment ionization. The sulfate and glucuronic acid moieties were located at C-3 and N-acetylglucosamine at C-20. An alternative localization of GlcNAc at C-21 of 5alpha-pregnane-3alpha,20alpha,21-triol cannot be excluded. Judging from the enzymatic hydrolysis of the conjugates, the sugar was attached in beta-glycosidic linkage. The mean excretion of N-acetylglucosaminides of the pregnanediols and pregnanetriols was 32.2 micromol/g creatinine (range 17.9-49.1 micromol) in five healthy women in the 38th-39th week of pregnancy. The mean excretion of 5beta-pregnane-3alpha,20alpha-diol glucuronide in the same women was 71 micromol/g creatinine, (range 27-127 micromol). This indicates that conjugation with N-acetylglucosamine constitutes a quantitatively important pathway of progesterone metabolism in human pregnancy.

In recent years electrospray has become the most popular ionization technique for the analysis of biomolecules. In the present study, we have compared the electrospray and continuous-flow fast-atom bombardment methods of sample introduction and ionization for the analysis of sulphated and sulphonated lipids by tandem mass spectroscopy. The tandem mass spectra obtained when using the two ionization techniques were similar; however, small differences were evident. These differences indicate that the precursor ions generated by fast-atom bombardment and electrospray have different internal energies. The two ionization techniques give similar sensitivities for the recording of collision-induced dissociation spectra. Complete structural information can be obtained from 5 to 50 ng of lipid

Dolichols and polyprenols are polyisoprenoid lipids found in all cells. Polyisoprenoids have recently been found covalently bound to cellular proteins constituting a new type of post-translational modification. To study these compounds effectively in biological systems a sensitive mass spectrometric procedure giving molecular weight and structural information is required. In the present study an assessment has been made of possible mass spectrometric procedures. Dolichols and polyprenols have been analysed at the pmol level in their underivatized form and as tert-butyldimethylsilyl ethers by electron ionization mass spectrometry. Sulphated dolichols and polyprenols have been analysed at a pmol level by fast-atom bombardment and at the sub-pmol level by electrospray mass spectrometry. Dolichol phosphates have also been analysed by electrospray mass spectrometry. Collision-induced dissociation spectra of the underivatized and derivatized polyisoprenoids have been recorded. These spectra provide structural information from only pmols of sample.

Collision-induced dissociation (CID) mass spectra were recorded on underivatized, hydroxylated lipoxygenase products, including S(S)-, 12(S)- and 15(S)-hydroxyeicosatetraenoic adds (HETE), leukotriene (LT) B4, LTB5, the LTB4 metabolites 20-OH-LTB4, 20-COOH-LTB4 and 10,11-dihydro-LTB4, the LTB4 isomers 6-trans-LTB4, 6-trans,12-epi-LTB4 and S(S),12(S)-dihydroxyeicosatetraenoic acid (diHETE) as well as S(S),6(R)-diHETE, 14(S), 15(R)-diHETE and lipoxin (LX) A4 and B4. Pseudomolecular [M-H]- parent ions were generated by electrospray ionization. The ions were accelerated to 4 keV, selected by the double focusing sectors Of 2 hybrid tandem mass spectrometer, decelerated to 400 eV and focused into a collision cell containing xenon. The resulting fragment ions were mass analysed by an orthogonal time-of-flight analyser. The CID spectra obtained were structurally informative allowing isomer differentiation.

The metabolism of 27-hydroxycholesterol and 25-hydroxycholesterol was studied in cultures of human diploid fibroblasts. Both steroids underwent 7 alpha-hydroxylation with subsequent oxidation to 7 alpha-hydroxy-3-oxo-delta 4 steroids. A minor fraction of the 27-hydroxysteroids was oxidized to acids. Competition experiments indicated that both hydroxycholesterols were hydroxylated by the same enzyme, different from cholesterol 7 alpha-hydroxylase. 7 alpha,25-Dihydroxycholesterol suppressed the activity of HMG-CoA reductase at least as effectively as 25-hydroxycholesterol whereas 7 alpha,25-dihydroxy-4-cholesten-3-one was a less effective suppressor. The results suggest that cholesterol might be converted to 7 alpha-hydroxylated bile acid precursors in extrahepatic tissues in vivo and that the regulation of the activity of HMG-CoA reductase by oxysterols might be modulated by 7 alpha-hydroxylation and subsequent oxidation by 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase/isomerase.

A total of 100 nonobese and normolipidemic subjects (29 control subjects, 49 patients with cholesterol stones [CSs], and 22 patients with brown pigment stones) were studied to elucidate the pathogenetic contributions of deoxycholate (DC) to supersaturated bile formation with special reference to de novo syntheses of cholesterol and bile acids in the liver. A higher proportion of DC was observed in gallbladder bile from patients with CSs (CSs; 21.7 ± 1.4%, mean ± SEM, vs. control subjects; 10.2 ± 0.9%). Cholesterol saturation in bile was elevated parallel to the increase of DC (r = .48; P = .0002), irrespective of the existence of stones. In a comparison between the 52 subjects with increased DC in bile (>10% of biliary bile acids) and the 20 subjects without the increase (

The effect of bile acid precursors on the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was investigated. Cholesterol and 34 of its derivatives, including 23 potential intermediates in bile acid biosynthesis, were incubated with cultures of human fibroblasts for 24 h in the absence or presence of lipoproteins, and the activity of HMG-CoA reductase was then determined. In the absence of lipoproteins, many of the bile acid intermediates were inhibitory at a high concentration (2.5 microM), while only three, 27-hydroxycholesterol, 7 alpha, 27-dihydroxy-4-cholesten-3-one, and 7 alpha, 12 alpha, 27-trihydroxy-4-cholesten-3-one, caused a significant suppression at lower concentrations (often > 80% suppression at 0.25 microM). Even at 0.06 microM these sterols caused > 50% suppression of the enzyme activity. In addition, 27-hydroxy-4-cholesten-3-one, not usually considered to be an intermediate in bile acid biosynthesis, was a very potent inhibitor. Comparative studies showed that the effect of the three bile acid precursors was similar to that of 25-hydroxy-, 24-hydroxy-, and 7-oxo-cholesterol and 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one. The presence of lipoproteins decreased or eliminated the inhibitory effect of most intermediates. Studies of the metabolism of the three most potent inhibitors in the fibroblasts indicated that the suppression was due to the compounds per se and not to products of their metabolism. The results show that a few specific intermediates in the formation of bile acids are potent suppressors of HMG-CoA reductase.(ABSTRACT TRUNCATED AT 250 WORDS)

Liver X receptors (Lxrα and Lxrβ) are ligand-dependent nuclear receptors critical for ventral midbrain neurogenesis in vivo. However, no endogenous midbrain Lxr ligand has so far been identified. Here we used LC/MS and functional assays to identify cholic acid as a new Lxr ligand. Moreover, 24(S),25-epoxycholesterol (24,25-EC) was found to be the most potent and abundant Lxr ligand in the developing mouse midbrain. Both Lxr ligands promoted neural development in an Lxr-dependent manner in zebrafish in vivo. Notably, each ligand selectively regulated the development of distinct midbrain neuronal populations. Whereas cholic acid increased survival and neurogenesis of Brn3a-positive red nucleus neurons, 24,25-EC promoted dopaminergic neurogenesis. These results identify an entirely new class of highly selective and cell type-specific regulators of neurogenesis and neuronal survival. Moreover, 24,25-EC promoted dopaminergic differentiation of embryonic stem cells, suggesting that Lxr ligands may thus contribute to the development of cell replacement and regenerative therapies for Parkinson's disease.

Fast atom bombardment mass spectrometry was used as a detection method in a study of the extraction of urinary bile acids with octadecylsilane-bonded silica. The procedure commonly used in many laboratories was found to result in significant losses of sulfated taurine-conjugated bile acids. Losses of other double conjugates and of bile acid and bile alcohol glucuronides were also seen. The losses were avoided by addition of an equal volume of 0.5 M triethylamine sulfate to the urine before passing it through the sorbent bed. Quantitative elution was then achieved with methanol. Batch variations were observed with sorbents from two different manufacturers.

In order to establish the position of conjugation of bile acids with glucose or N-acetylglucosamine, glucosides of chenodeoxycholic and hyodeoxycholic acids and of 13C-labeled cholic, lithocholic, chenodeoxycholic, hyodeoxycholic, and ursodeoxycholic acids, and N-acetylglucosaminides of ursodeoxycholic, isoursodeoxycholic, 3-dehydro-ursodeoxycholic, and ursodeoxycholylglycine were synthesized in vitro. The conjugates were purified by anion-exchange chromatography and reversed-phase HLPC and were analyzed by gas chromatography-mass spectrometry. The glucosides of chenodeoxycholic and hyodeoxycholic acids were also analyzed after periodate and chronic acid oxidation. All conjugates were analyzed by fast atom bombardment mass spectrometry with collision-induced dissociation. Glucose conjugation was shown to occur at C-3 in all bile acid glucosides studied. In contrast, the selective N-acetylglucosaminidation of 7 beta-hydroxy bile acids was shown to occur at the 7 beta-position.

When analyzed by fast atom bombardment mass spectrometry, taurine-conjugated bile acids give intense [M-H]-pseudomolecular ions that can be subjected to collision-induced fragmentation to give structural information. A method has been developed that permits rapid coupling of taurine to unconjugated, glycine-conjugated, sulfated, and glucuronidated bile acids. The reaction is performed for 2 h at room temperature in aqueous pyridine hydrochloride buffer, with or without dioxane, using 0.1 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as the coupling agent and 0.2 M taurine. The yields are higher than 95%. In contrast to published coupling reactions, the method permits conjugation of bile acids with the labile 7 alpha-hydroxy-3-oxo-4-ene structure.