Jan Sjövall

Pregnenolone and its sulfate ester in the rat brain

Pregnenolone (P) and its sulfate ester (PS) have been characterized in the brain of adult male rats. The concentration of P (38.4 +/- 6.9 and 22.1 +/- 2.9 ng/g, mean +/- S.D., in anterior and posterior brain, respectively) exceeded that of PS in brain (15.8 +/- 3.0 and 5.7 +/- 2.1 ng/g in the same fractions) and largely those of P and PS in plasma (1.3 +/- 0.2 and 1.4 +/- 0.3 ng/g, respectively). The level of P in brain was much larger than that of dehydroepiandrosterone sulfate (DS), characterized and measured previously (Corpéchot et al.). Brain P and PS levels did not seem to depend on steroidogenic gland secretion: no meaningful difference occurred in brain 15 days after adrenalectomy plus orchiectomy, compared with sham-operated controls. It is proposed that, as that of DS (ref. 5) P and PS formation or accumulation (or both) in the rat brain depend on in situ mechanisms unrelated to the peripheral endocrine gland system.

Polyunsaturated Fatty Acids Including Docosahexaenoic and Arachidonic Acid Bind to the Retinoid X Receptor α Ligand-binding Domain

Nuclear receptors (NRs) constitute a large and highly conserved family of ligand-activated transcription factors that regulate diverse biological processes such as development, metabolism, and reproduction. As such, NRs have become important drug targets, and the identification of novel NR ligands is a subject of much interest. The retinoid X receptor (RXR) belongs to a subfamily of NRs that bind vitamin A metabolites (i.e. retinoids), including 9-cis-retinoic acid (9-cis-RA). However, although 9-cis-RA has been described as the natural ligand for RXR, its endogenous occurrence has been difficult to confirm. Recently, evidence was provided for the existence of a different natural RXR ligand in mouse brain, the highly enriched polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) (Mata de Urquiza et al. (2000) Science 290, 2140–2144). However, the results suggested that supra-physiological levels of DHA were required for efficient RXR activation. Using a refined method for ligand addition to transfected cells, the current study shows that DHA is a more potent RXR ligand than previously observed, inducing robust RXR activation already at low micromolar concentrations. Furthermore, it is shown that other naturally occurring PUFAs can activate RXR with similar efficiency as DHA. In additional experiments, the binding of fatty acid ligands to RXRα is directly demonstrated by electrospray mass spectrometry of the noncovalent complex between the RXR ligand-binding domain (LBD) and its ligands. Data is presented that shows the noncovalent interaction between the RXR LBD and a number of PUFAs including DHA and arachidonic acid, corroborating the results in transfected cells. Taken together, these results show that RXR binds PUFAs in solution and that these compounds induce receptor activation, suggesting that RXR could function as a fatty acid receptor in vivo.

The multicomponent analysis of conjugates of neutral steroids in urine by lipophilic ion exchange chromatography and computerised gas chromatography-mass spectrometry

Abstract A method for the separation and analysis of conjugates of neutral steroids in urine is described. Following Amberlite XAD-2 extraction of steroids, separation into neutral, glucuronide, monosulphate and disulphate conjugate groups was achieved by ion exchange chromatography on the lipophilic gel, diethylaminohydroxypropyl Sephadex LH-20 (DEAP-LH-20). Enzymatic and solvolytic procedures were used to hydrolyse the conjugate moiety and the steroids were analysed by gas-chromatography using open-tubular glass capillary columns after the preparation of the O-methyloxime-trimethylsilyl ether derivative. Characterization of the steroids was made by computerised GC-MS following repetitive magnetic scanning? Examples of the application of the method to the analysis of urine from normal male subjects, and a patient with Cushings syndrome are presented.

Importance of a Novel Oxidative Mechanism for Elimination of Intracellular Cholesterol in Humans

We have recently demonstrated that cultured human alveolar macrophages efficiently convert cholesterol into excretable 27-oxygenated products. We show here that increasing the intracellular concentration of cholesterol by a factor of 10 leads to about a twofold increase in the excretion of 27-oxygenated products from cultured macrophages. Inhibition of the sterol 27-hydroxylase caused a significant intracellular accumulation of cholesterol. A direct comparison was made between flux of cholesterol and 27-oxygenated products from macrophages preloaded with [4-14C]cholesterol. Under the specific conditions employed with fetal calf serum in the culture medium, the flux of 27-oxygenated products was about 10% of that of cholesterol. Since the sterol 27-hydroxylase, which converts cholesterol to 27-oxygenated products, is present in many cell types, we suggest that 27-oxygenation is a general mechanism for removal of intracellular cholesterol. To evaluate this hypothesis, we measured the net uptake by the human liver of circulating 27-oxygenated products, which was found to be about 20 mg/24 h. This uptake corresponds to approximately 4% of the bile acid production, assuming quantitative conversion into bile acids. It is concluded that the 27-hydroxylase pathway is of significance for elimination of extrahepatic cholesterol.

Validation of an analytical procedure to measure trace amounts of neurosteroids in brain tissue by gas chromatography–mass spectrometry

A selective and extremely sensitive procedure has been developed and optimized, using high-performance liquid chromatography (HPLC), specific derivatization and gas chromatography-mass spectrometry (GC-MS), to simultaneously quantify very small amounts of different neurosteroids from rat brain. Unconjugated and sulfated steroids in brain extracts were separated by solid-phase extraction. The unconjugated fraction was further purified by HPLC, the steroids being collected in a single fraction, and the sulfated fraction was solvolyzed. All steroids were derivatized with heptafluorobutyric acid anhydride and analyzed by GC-MS (electron impact ionization) using selected-ion monitoring. High sensitivity and accuracy were obtained for all steroids. The detection limits were 1 pg for pregnenolone (PREG), dehydroepiandrosterone (DHEA) and their sulfate esters PREG-S and DHEA-S, 2 pg for progesterone (PROG) and 5 pg for 3alpha,5alpha-tetrahydroprogesterone (3alpha,5alpha-THP). In a pilot study on a rat brain, the concentrations of PREG-S and DHEA-S were 8.26+/-0.80 and 2.47+/-0.27 ng/g, respectively. Those of PREG, DHEA and PROG were 4.17+/-0.22, 0.45+/-0.02 and 1.95+/-0.10 ng/g, respectively. Good linearity and accuracy were observed for each steroid. The methodology validated here, allows femtomoles of neurosteroids, including the sulfates, found in small brain samples (at least equal to 10 mg) to be quantified simultaneously.

Separation and computerized gas chromatography-mass spectrometry of unconjugated neutral steroids in plasma

A method is described for analysis of unconjugated neutral steroids in plasma. It is based on extraction of the sample with Amberlite XAD-2 at elevated temperature, purification of the extract on lipophilic ion exchanging and neutral Sephadex derivatives, and gas chromatography-mass spectrometry-computer analysis of O-methyloxime-trimethylsilyl ether derivatives purified on hydrophobic Sephadex. Examples of the application of the method to analysis of steroids in plasma from pregnant women are given.

Bile acids and progesterone metabolites in intrahepatic cholestasis of pregnancy.

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 3α,5α 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.

Familial giant cell hepatitis associated with synthesis of 3 beta, 7 alpha-dihydroxy-and 3 beta,7 alpha, 12 alpha-trihydroxy-5-cholenoic acids.

Urinary bile acids from a 3-mo-old boy with cholestatic jaundice were analyzed by ion exchange chromatography and gas chromatography-mass spectrometry (GC-MS). This suggested the presence of labile sulfated cholenoic acids with an allylic hydroxyl group, a conclusion supported by analysis using fast atom bombardment mass spectrometry (FAB-MS). The compounds detected by FAB-MS were separated by thin layer chromatography and high performance liquid chromatography. The sulfated bile acids could be solvolyzed in acidified tetrahydrofuran, and glycine conjugates were partially hydrolyzed by cholylglycine hydrolase. Following solvolysis, deconjugation, and methylation with diazomethane, the bile acids were identified by GC-MS of trimethylsilyl derivatives. The major bile acids in the urine were 3 beta,7 alpha-dihydroxy-5-cholenoic acid 3-sulfate, 3 beta,7 alpha,12 alpha-trihydroxy-5-cholenoic acid monosulfate, and their glycine conjugates. Chenodeoxycholic acid and cholic acid were undetectable in urine and plasma. The family pedigree suggested that abnormal bile acid synthesis was an autosomal recessive condition leading to cirrhosis in early childhood.

Determination of steroid mono- and disulfates in human plasma.

Abstract A method for the determination of androsterone, dehydroepiandrosterone, epiandrosterone, 5α-androstane-3α,17β-diol, androst-5-ene-3β17α-diol, androst-5-ene-3β,17β-diol, pregnenolone, pregn-5-ene-3β,20α-diol and pregn-5-ene-3β,17α,20α-triol monosulfates and androst-5-ene-3β,17α-diol, androst-5-ene-3β,17β-diol and pregn-5-ene-3β,20α-diol disulfates in human plasma is described. A plasma extract is chromatographed on Sephadex LH-20 and a monosulfate and a disulfate fraction are obtained. After solvolysis, the steroids are purified on a silicic acid column and their trimethylsilyl ethers are analyzed by gas chromatography on QF-1 and SE-30 columns. The specificity of the method was established by mass spectrometric analysis of the effluent from the GLC columns. Concentrations of steroid mono- and disulfates in peripheral plasma of a group of young healthy female and male subjects are given.

Paracrine glucocorticoid activity produced by mouse thymic epithelial cells

Previous data have suggested that glucocorticoids (GCs) are involved in the differentiation of thymocytes into mature T cells. In this report we demonstrate that the mouse thymic epithelial cells (TEC) express the cytochrome P450 hydroxylases Cyp11A1, Cyp21, and Cyp11B1. These enzymes, in combination with 3β‐hydroxysteroid dehydrogenase (3βHSD), convert cholesterol into corticosterone, the major GC in rodents. In addition, when TEC were cocultured with ‘reporter cells’ containing the glucocorticoid receptor (GR) and a GR‐dependent reporter gene, a specific induction of reporter gene activity was observed. Induction of reporter gene activity was blocked when the TEC and reporter cells were incubated in the presence of the Cyp11B1 inhibitor metyrapone or the 3βHSD inhibitor trilostane, as well as by the GR antagonist RU486. Coculturing of TEC with thymocytes induced apoptosis in the latter, which was partially blocked by the enzyme inhibitors and RU486. We conclude that TEC secrete a GC hormone activity and suggest a paracrine role for this in thymocyte development.—Pazirandeh, A., Xue, Y., Rafter, I., Sjövall, J., Jondal, M., Okret, S. Paracrine glucocorticoid activity produced by mouse thymic epithelial cells. FASEB J. 13, 893–901 (1999)