Clostridium scindens American Type Culture Collection 35704 is capable of converting primary bile acids to toxic secondary bile acids, as well as converting glucocorticoids to androgens by side-chain cleavage. The molecular structure of the side-chain cleavage product of cortisol produced by C. scindens was determined to be 11β-hydroxyandrost-4-ene-3,17-dione (11β-OHA) by high-resolution mass spectrometry, 1H and 13C NMR spectroscopy, and X-ray crystallography. Using RNA-Seq technology, we identified a cortisol-inducible (∼1,000-fold) operon (desABCD) encoding at least one enzyme involved in anaerobic side-chain cleavage. The desC gene was cloned, overexpressed, purified, and found to encode a 20α-hydroxysteroid dehydrogenase (HSDH). This operon also encodes a putative “transketolase” (desAB) hypothesized to have steroid-17,20-desmolase/oxidase activity, and a possible corticosteroid transporter (desD). RNA-Seq data suggests that the two-carbon side chain of glucocorticords may feed into the pentose-phosphate pathway and are used as a carbon source. The 20α-HSDH is hypothesized to function as a metabolic “rheostat” controlling rates of side-chain cleavage. Phylogenetic analysis suggests this operon is rare in nature and the desC gene evolved from a gene encoding threonine dehydrogenase. The physiological effect of 11β-OHAD on the host or other gut microbes is currently unknown.

Oxysterols constitute a class of cholesterol derivatives that exhibit broad biological effects ranging from cytotoxicity to regulation of nuclear receptors. The role of oxysterols such as 7-ketocholesterol (7-KC) in the development of retinal macular degeneration and atheromatous lesions is of particular interest, but little is known of their metabolic fate. We establish that the steroid/sterol sulfotransferase SULT2B1b, known to efficiently sulfonate cholesterol, also effectively sulfonates a variety of oxysterols, including 7-KC. The cytotoxic effect of 7-KC on 293T cells was attenuated when these cells, which do not express SULT2B1b, were transfected with SULT2B1b cDNA. Importantly, protection from 7-KC-induced loss of cell viability with transfection correlated with the synthesis of SULT2B1b protein and the production of the 7-KC sulfoconjugate (7-KCS). Moreover, when 7-KCS was added to the culture medium of 293T cells in amounts equimolar to 7-KC, no loss of cell viability occurred. Additionally, MCF-7 cells, which highly express SULT2B1b, were significantly more resistant to the cytotoxic effect of 7-KC. We extended the range of oxysterol substrates for SULT2B1b to include 7a/ 7b-hydroxycholesterol and 5a,6a/5b,6b-epoxycholesterol as well as the 7a-hydroperoxide derivative of cholesterol. Thus, SULT2B1b, by acting on a variety of oxysterols, offers a potential pathway for modulating in vivo the injurious effects of these compounds.-Fuda, H., N. B. Javitt, K. Mitamura, S. Ikegawa, and C. A. Strott. Oxysterols are substrates for cholesterol sulfotransferase. J. Lipid Res.

Salt-inducible kinase 3 (SIK3), an AMP-activated protein kinase-related kinase, is induced in the murine liver after the consumption of a diet rich in fat, sucrose, and cholesterol. To examine whether SIK3 can modulate glucose and lipid metabolism in the liver, we analyzed phenotypes of SIK3-deficent mice. Sik3 −/− mice have a malnourished the phenotype (i.e., lipodystrophy, hypolipidemia, hypoglycemia, and hyper-insulin sensitivity) accompanied by cholestasis and cholelithiasis. The hypoglycemic and hyper-insulin-sensitive phenotypes may be due to reduced energy storage, which is represented by the low expression levels of mRNA for components of the fatty acid synthesis pathways in the liver. The biliary disorders in Sik3 −/− mice are associated with the dysregulation of gene expression programs that respond to nutritional stresses and are probably regulated by nuclear receptors. Retinoic acid plays a role in cholesterol and bile acid homeostasis, wheras ALDH1a which produces retinoic acid, is expressed at low levels in Sik3 −/− mice. Lipid metabolism disorders in Sik3 −/− mice are ameliorated by the treatment with 9-cis-retinoic acid. In conclusion, SIK3 is a novel energy regulator that modulates cholesterol and bile acid metabolism by coupling with retinoid metabolism, and may alter the size of energy storage in mice.

Bile acids, synthesized from cholesterol by hepatic enzymes, are excreted into the duodenum via the bile duct as their glycine and taurine conjugates. After assisting in the lipolysis and absorption of fats in the intestinal lumen, bile acids are then returned to the liver. According to this enterohepatic circulation, very small quantities of bile acids are present in blood of healthy subjects. However, in hepatobiliary diseases, disturbances in their synthesis and clearance by the liver, as well as absorption by the intestines cause changes in the level of not only bile acids but also their sulfates and glucuronides in the blood and urine. 1 Since the first report of the occurrence of the bile acid glucuronide in human urine, it has been believed that bile acids are conjugated with glucuronic acid through the inherent 3α -hydroxy group on the steroid nucleus to form their ether type glucuronides. 2-6 Recently, we have disclosed the formation of ester-type acyl glucuronides of bile acid conjugated through the 24-carboxy group in an incubation mixture with hepatic microsomal fractions from rats 7 , employing liquid chromatography (LC)/atmospheric pressure chemical ionization (APCI)-mass spectrometry (MS); here the acyl glucuronides were characterized as their acetate-methyl ester derivatives. In addition, we have synthesized the acyl glucuronides of cholic, chenodeoxycholic, deoxycholic, ursodeoxycholic and lithocholic acids as authentic specimens. 8 The present paper deals with the development of the simultaneous determination of the bile acid 24-glucuronides in human urine using LC/electrospray ionization (ESI)-MS without derivatization. Experimental LC/ESI-MSThe liquid chromatographic separation was performed with a Nano-space SI-1 (Shiseido, Ltd., Tokyo, Japan) on a Develosil ODS-HG-5 column (5 µm, 150×2.0 mm i.d.) (Nomura Chemical, Aichi, Japan) with a flow rate of 150 µl/min. The mass spectrometric detection was carried out using a JMS-LCmate doublefocusing mass spectrometer (JEOL, Tokyo, Japan) equipped with an ESI system in the negative ion mode. The resolution of the mass spectrometer was set at 750 and 5000 for the low-resolution and high-resolution analyses, respectively. For the rock-mass signal at m/z 602.9125, the standard solution containing sodium trifluoroacetic acid and sodium formic acid (each 50 µg/ml) in MeOH was added to the postcolumn effluent using a "T-type" connector at a flow rate of 50 µl/min. MaterialsBile acid 3-9 and 24-glucuronides 8 (Fig. 1) A method for the separation and determination of bile acid 24-glucuronides using liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometry (MS) has been developed. In the ESI mode, 24-glucuronides were characterized by an intense deprotonated molecule [M-H] -with a fragment ion [M-H-176] -formed by elimination of the glucuronic acid moiety. The ratio of these negative ions was significantly influenced by the orifice-1 and the ring lens voltages. Bile acid 24-glucuronides in human urine were extracted with an OASIS HLB car...

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