Secondary bile acid[

Lithocholic acid is a secondary bile acid that is toxic[1].

The pregnane X receptor (PXR) is the molecular target for catatoxic steroids such as pregnenolone 16α-carbonitrile (PCN), which induce cytochrome P450 3A (CYP3A) expression and protect the body from harmful chemicals. In this study, we demonstrate that PXR is activated by the toxic bile acid lithocholic acid (LCA) and its 3-keto metabolite. Furthermore, we show that PXR regulates the expression of genes involved in the biosynthesis, transport, and metabolism of bile acids including cholesterol 7α-hydroxylase (Cyp7a1) and the Na+-independent organic anion transporter 2 (Oatp2). Finally, we demonstrate that activation of PXR protects against severe liver damage induced by LCA. Based on these data, we propose that PXR serves as a physiological sensor of LCA, and coordinately regulates gene expression to reduce the concentrations of this toxic bile acid. These findings suggest that PXR agonists may prove useful in the treatment of human cholestatic liver disease [1].

Maintenance and Treatment of PXR−/− and Control Mouse Populations. [1]
Adult male wild-type (PXR+/+) and PXR-null (PXR−/−) mice were maintained on standard laboratory chow and were allowed food and water ad libitum. Mice were treated with PCN (0.4 mg/g), dexamethasone (0.1 mg/g), sodium phenobarbital (PB, 0.1 mg/g), or LCA (0.125 mg/g). All inducers were dissolved in corn oil and injected intraperitoneally either once (PCN, dexamethasone, and PB) or twice (LCA) a day for 4 days. Animals were killed 24 h following the final injection. For LCA and PCN cotreatment, PCN was administered by gavage (0.5 mg/g in corn oil) for 3 days. Subsequently, PCN treatment was continued for a further 4 days, during which time animals also received a daily i.p. injection of LCA (0.25 mg/kg in corn oil). Mice were exsanguinated 24 h following the final injection and serum alanine transaminase (ALT) and sorbitol dehydrogenase (SDH) levels determined by using standard techniques. Livers were fixed in neutral-buffered formalin solution Sigma, embedded in paraffin wax, and stained with hematoxylin and eosin. Differences between SDH and ALT levels in vehicle- and LCA-treated and PCN- and LCA-treated animals were determined by using a one-way ANOVA. Significant differences were determined by using Duncan's multiple range post hoc test.

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Quantitation of LCA in Urine. [1]
LCA levels in urine were quantitated by atmospheric pressure ionization–liquid chromatography mass spectrometry (API-LCMS). Briefly, an equal volume of mouse urine and a 5 μg/ml methanolic solution of 2,2,4,4-d4-cholic acid (D4-cholic acid) were combined. Samples were sonicated, centrifuged (3,000 × g for 10 min), and filtered through a 0.45-μm filter unit before injection onto the analytical column of an LCMS instrument. LCA and D4-cholic acid were detected as the molecular ions ([M-H−]) 375 and 311 m/z, respectively, in the negative selected ion monitoring mode of the instrumentation. LCA concentrations in the study samples were calculated by comparison to standard solutions of LCA containing D4-cholic acid as internal standard. The significance of differences between mean values was analyzed by using an unpaired Student's t test.

[1]. The nuclear receptor PXR is a lithocholic acid sensor that protects against liver toxicity. Proc Natl Acad Sci U S A. 2001;98(6):3369-3374.

Lithocholic acid-2,2,4,4-d4 is a deuterated compound and a lithocholic acid.

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It has been almost 30 years since Hans Selye first showed that PCN treatment blocks the hepatotoxicity and mortality caused by LCA treatment in rats. In this report, we have used PXR-null mice to demonstrate that the orphan nuclear receptor PXR mediates the hepatoprotective effects of PCN against LCA-induced toxicity. Moreover, we have shown that PXR is activated by LCA and its 3-keto metabolite and coordinately regulates genes involved in the biosynthesis, transport, and metabolism of LCA. Our results indicate that PXR plays a fundamental role in protecting the liver against pathophysiological levels of LCA. PXR thus joins FXR as nuclear receptors that are activated by bile acids. Although PXR and FXR are activated by distinct sets of bile acids, both receptors are activated by LCA. This raises the interesting possibility that PXR and FXR cooperate to remove LCA from the body when its concentrations reach pathophysiological levels.
Several previous reports suggest that our findings may have implications in the treatment of human cholestatic liver disease. Notably, urine from patients suffering from cholestasis contains elevated levels of 6-hydroxylated bile acids (including the LCA metabolite hyodeoxycholic acid), which are products of CYP3A4. These findings suggest that increased 6-hydroxylation is a relevant mechanism for reducing the levels of toxic bile acids in humans. Elevated levels of 6-hydroxylated bile acids are also observed in the urine of healthy subjects treated with the PXR ligand rifampicin. Interestingly, rifampicin has been used successfully in the treatment of pruritus associated with intrahepatic cholestasis and, in some instances, has been reported to induce remission of cholestasis. The molecular basis for these clinical effects has remained obscure. Based on our data, we suggest that the anticholestatic effects of rifampicin may be mediated through PXR, and that potent PXR ligands may be more efficacious in the treatment of cholestasis, a severe hepatic disease for which there is no known cure. [1]

C24H36D4O3

380.60

380.323

83701-16-0

Lithocholic acid;434-13-9

16217405

White to off-white solid powder

1.085 g/cm3

510.992ºC at 760 mmHg

184-186ºC(lit.)

276.926ºC

5.507

2

3

4

27

574

9

[2H]C1(C[C@@]2([C@H]3CC[C@]4([C@H]([C@@H]3CC[C@@H]2C([C@@H]1O)([2H])[2H])CC[C@@H]4[C@H](C)CCC(=O)O)C)C)[2H]

SMEROWZSTRWXGI-POXZWENPSA-N

InChI=1S/C24H40O3/c1-15(4-9-22(26)27)19-7-8-20-18-6-5-16-14-17(25)10-12-23(16,2)21(18)11-13-24(19,20)3/h15-21,25H,4-14H2,1-3H3,(H,26,27)/t15-,16-,17-,18+,19-,20+,21+,23+,24-/m1/s1/i10D2,14D2

(4R)-4-[(3R,5R,8R,9S,10S,13R,14S,17R)-2,2,4,4-tetradeuterio-3-hydroxy-10,13-dimethyl-3,5,6,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoic acid

83701-16-0; Lithocholic acid-2,2,4,4-d4; Lithocholic Acid-d4; (4R)-4-[(3R,5R,8R,9S,10S,13R,14S,17R)-2,2,4,4-tetradeuterio-3-hydroxy-10,13-dimethyl-3,5,6,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoic acid; Lithocholic-2,2,4,4-d4 acid; Lithocholic Acid D4; Cholan-24-oic-2,2,4,4-d4 acid, 3-hydroxy-, (3a,5ss)-; (3a,5ss)-3-Hydroxycholan-24-oic-2,2,4,4-d4 acid; Lithocholic acid-d4; Lithocholic Acid-2,2,4,4-d4;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 50 mg/mL (131.37 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (13.14 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 5 mg/mL (13.14 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)