VDR/vitamin D receptor; PXR; FXR; Microbial Metabolite; Endogenous Metabolite

Lithocholic Acid has an IC50 of 0.7 μM and 1.4 μM, respectively, to block FXR activation produced by GW4064 and CDCA [5]. In HepG2 cells, 100 nM GW4064-induced BSEP expression is inhibited by 10-30 μM lithocholic acid over a 24-hour period [5]. Lithocholic Acid (0-500 μM) suppresses neuroblastoma cell growth (BE(2)-m17, SK-n-SH, SK-n-MCIXC, and Lan-1) in a dose-dependent manner[3].

Lithocholic Acid (LCA) as an Effective Cholestasis Inducer: Mechanisms and Modeling Methods
Pathogenic Mechanisms
• As a toxic secondary bile acid, LCA induces intrahepatic cholestasis by altering hepatocyte membrane composition and bile secretion function.
• Its toxic effects include promoting biliary epithelial cell injury, inflammatory cell infiltration, and bile acid metabolism disorders.
• It exacerbates cholestasis by activating nuclear receptor pathways (e.g., FXR/PXR).

Animal Model Construction
Rat Model (Acute Cholestasis)
Strain: Male Wistar rats (250–300 g)
Dosing Protocol:
Dose: 0.2 μmol/100 g
Route: Intravenous injection (dissolved in 7.5% bovine serum albumin + 0.45% saline)
Sacrifice Time: 1 hour post-administration

Mouse Model (Subacute Cholestasis)
Strain: Male ICR mice (5–7 weeks old)
Dosing Protocol:
Dose: 150 mg/kg
Route: Oral gavage (dissolved in corn oil)
Frequency: Twice daily for 5 administrations
Sacrifice Time: 12 hours after the last dose

Evaluation Parameters
Serum Biochemistry:
• Significant increases in total bilirubin (TBIL) and direct bilirubin (DBIL)
• Elevated liver enzyme activities (ALT, AST, ALP)

Histopathology:
• Hepatocyte vacuolar degeneration and bile duct proliferation
• Inflammatory cell infiltration in portal areas
• Bile capillary cholestasis

Functional Changes:
• Transient increase followed by a significant decrease in bile flow
• Disrupted bile acid (TBA) metabolism

Key Considerations
• Strictly control IV concentration to prevent hemolysis.
• Administer oral doses at fixed light-cycle timepoints to minimize circadian rhythm effects.
• Include vehicle control groups (bovine serum albumin/corn oil) to exclude solvent interference.

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When added to the food at a rate of 0.6% for seven days, lithocholic acid raises the levels of TGFB1, TGFBR1, and TGFBR2 mRNA in the liver of male C57BL/6 mice, activates SMAD3, and causes biliary injury [4]. Male C57BL/6 mice given intraperitoneal injections of lithocholic acid (125 mg/kg, twice daily for four days) develop liver damage and have elevated levels of AST, ALT, and ALP [2].

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The cholestatic liver damage was generated by alphanaphthyl isothiocyanate (ANIT), 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and Lithocholic acid (LCA). The results indicated that the levels of bile acids were commonly increased in plasma of three mouse cholestasis models, while arginine was decreased. The level of plasma glutathione was decreased in ANIT- and LCA-induced intrahepatic PBC and PSC, respectively. But, the liver glutathione was decreased in DDC induced extrahepatic PSC. The level of plasma phospholipids was elevated in ANIT and DDC models, whereas that was depleted in LCA model. And protoporphyrin IX was significantly increased in the liver of DDC model. These metabolomics data could potentially distinguish the metabolic differences of three types of cholestasis, contributing to the understanding of the potential mechanism of cholestatic liver damage. [2]

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Lithocholic acid (LCA) induced expression of TGFB1 and the receptors TGFBR1 and TGFBR2 in the liver. In addition, immunohistochemistry revealed higher TGFβ expression around the portal vein after Lithocholic acid/LCA exposure and diminished SMAD3 phosphorylation in hepatocytes from Smad3-null mice. Serum metabolomics indicated increased bile acids and decreased lysophosphatidylcholine (LPC) after LCA exposure. Interestingly, in Smad3-null mice, the metabolic alteration was attenuated. LCA-induced lysophosphatidylcholine acyltransferase 4 (LPCAT4) and organic solute transporter β (OSTβ) expression were markedly decreased in Smad3-null mice, whereas TGFβ induced LPCAT4 and OSTβ expression in primary mouse hepatocytes. In addition, introduction of SMAD3 enhanced the TGFβ-induced LPCAT4 and OSTβ expression in the human hepatocellular carcinoma cell line HepG2. In conclusion, considering that Smad3-null mice showed attenuated serum ALP activity, a diagnostic indicator of cholangiocyte injury, these results strongly support the view that TGFβ-SMAD3 signaling mediates an alteration in phospholipid and bile acid metabolism following hepatic inflammation with the biliary injury[4].

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Lithocholic acid exposure enhanced TGFβ and the receptors mRNA level in the liver [4]
The influence of Lithocholic acid (LCA) exposure on TGFβ signaling was investigated using C57BL/6 mice treated with the synthetic AIN93G diet (Cont) and 0.6% LCA-supplemented AIN93G diet (LCA) for 7 days. Hepatic TGFB1, TGFBR1, and TGFBR2 mRNA levels increased after LCA exposure, although TGFBR3 mRNA level did not changed in the livers (Fig. 1). These results suggest that LCA exposure stimulates TGFβ signaling in the livers.

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LCA-induced liver injury was alleviated in Smad3-null mice [4]
TGFβ activates SMAD3 via the TGFβ receptors. Thus, to investigate whether SMAD3 was involved in Lithocholic acid/LCA-induced liver injury, Smad3-null mice were treated with control diet and LCA diet for 6 days. After LCA exposure, the liver mass of Smad3-null mice was smaller than that of LCA-treated wild-type mice (Fig. 2A). In addition, LCA-increased serum ALP activities were significantly attenuated in the Smad3-null mice, although serum ALT activities were not changed (Fig. 2B, C). Furthermore, liver histology showed mild features of inflammatory cell infiltration around the portal vein in Smad3-null mice, which was not observed in similarly treated wild-type mice (Fig. 2D). Immunohistochemistry revealed TGFβ protein around the portal vein with lower expression of the TGFβ in Smad3-null mice compared with wild-type mice (Fig. 2E), suggesting lower TGFβ stimulation of the SMAD3 activation in the liver (supplementary Fig. I). In addition, a dramatic attenuation of the SMAD3 phosphorylation signal was observed in the hepatocyte nuclei of Smad3-null mice (Fig. 2E). These results suggest that TGFβ-SMAD3 signaling is associated with the LCA-induced biliary injury and raise the possibility that TGFβ-SMAD3 signaling alters hepatic metabolism.

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Difference between wild-type and Smad3-null mice in the serum metabolome after Lithocholic acid/LCA exposure [4]
To examine serum metabolites, PLS and contribution analyses were performed with UPLC-ESI-QTOFMS negative mode data derived from serum of mice fed Lithocholic acid/LCA or control diet. PLS analysis showed a separation between the LCA-treated wild-type (Fig. 3A) and the LCA-treated Smad3-null group that was further examined with a loadings plot (Fig. 3B). Contribution analysis revealed 10 enhanced and 10 attenuated ions as the top-ranking ions giving rise to the separation. Lysophosphatidylcholine (LPC) and fatty acid fragments were determined as raised ions in Smad3-null mice compared with the wild-type mice (Table 1). The most lowered ions were derived from bile salts (Table 2). After LCA feeding, the serum metabolome of Smad3-null mice was much different from that of the wild-type mice in LPC and bile salts.

Treatment of cells with Lithocholic acid/LCA, hydrogen peroxide or z-DEVD-fmk [3]
Stock solutions of Lithocholic acid/LCA in varying concentrations were first made in 100% dimethyl sulfoxide (DMSO). For treatment of cultured human NB, human BC and rat GL cells, these stock solutions were then diluted to the indicated final concentration of LCA (the final concentration of DMSO was always kept at 1%) in either a 1:1 mixture of DMEM and Ham's F-12 Nutrient Mixture supplemented with 10% FBS (for SK-n-MCIXC and BE(2)-m17 cells), EMEM supplemented with 10% FBS (for SK-n-SH cells), DMEM supplemented with 10% BCS (for Lan-1 cells) or a 1:1 mixture of DMEM and Ham's F-12 Nutrient Mixture supplemented with 10% FBS and 1X anti-mycotic/anti-biotic (for MCF7 and F98 cells). For treatment of human primary neurons with LCA, stock solutions of LCA in varying concentrations made in 100% DMSO were diluted in EMEM containing 0.225% sodium bicarbonate, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1% dextrose and 5% FBS to the indicated final concentration of LCA (the final concentration of DMSO was always kept at 1%). For treatment of cultured cancer cells or primary neurons with LCA, they were incubated for 48 hours in the presence LCA at the indicated final concentrations; control cells were treated with an empty DMSO vehicle only. For cell treatment with hydrogen peroxide, its 30% stock solution was diluted in sterile H2O and added directly to the cell cultures after 24 h of their pre-treatment with LCA or an empty DMSO vehicle only; cells were then incubated for 24 h. In experiments involving cell treatment with z-DEVD-fmk, this caspase-3 inhibitor was added to a final concentration of 5 μM simultaneously with LCA or an empty DMSO vehicle only.

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Cell viability assays [3]
The number of viable cells in cultures exposed to Lithocholic acid/LCA was measured using the MTT (3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide)-based CellTiter 96 Non-Radioactive Cell Proliferation Assay. In this assay, only viable, metabolically active cells were able to reduce the yellow tetrazolium salt of MTT to form a purple formazan product. This insoluble product was solubilized by the addition of a detergent. The resulting intracellular purple formazan was then detected spectrophotometrically using a 96-well plate reader at a wavelength of 570 nm. The signal was corrected to account for cellular debris using a wavelength of 630 nm. Chromatin in cells exposed to LCA and/or hydrogen peroxide and/or z-DEVD-fmk was visualized with the fluorescent dye Hoechst used at a final concentration of 4 μM in culture media and viewed using fluorescence microscopy. For each cell culture, the percentage of viable non-apoptotic cells carrying intact, non-fragmented nuclei containing non-condensed chromatin was calculated. Dead apoptotic cells carried fragmented nuclei containing condensed chromatin, a hallmark event of apoptotic death.

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Visualization of mitochondria and measurement of the mitochondrial membrane potential by fluorescence microscopy [3]
Mitochondrial morphology of cells treated with Lithocholic acid/LCA was visualized using MitoTracker Red CMXRos used at a concentration of 125 nM in the culture media. Cells were viewed using fluorescence microscopy, and the percentage of cells displaying fragmented mitochondria was calculated. The mitochondrial membrane potential (∆Ψ) was measured using tetramethylrhodamine ethyl ester (TMRE), a cell-permeant, cationic fluorescent dye. The extent of reversible sequestration of TMRE by mitochondria is proportional to the value of ∆Ψ. Cells were incubated with 50 nM TMRE for 20 min and directly viewed using fluorescence microscopy. The percentage of TMRE-positive cells displaying a detectable level of ∆Ψ was calculated.

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Transient Transfection Assay [5]
HepG2 cells were seeded at a density of 3.2 × 104 cells/well of 96-well plates in DMEM containing 10% FBS 24 h prior to transfection. Cells were transfected with transfection mixes in serum-free Opti-MEM I medium using the FuGENE6 transfection reagent according to the manufacturer's instructions. Typically, transfection mixes for each well contained 0.405 μl of FuGENE6, 3 ng of pcDNA3.1-GAL4-hFXR (LBD) expression vector, 3 ng of pcDNA3.1-hRXRα expression construct, and 60 ng of pUAS(5X)-tk-LUC reporter vector and 60 ng of pCMV-lacZ as an internal control for transfection efficiency. Cells were incubated in the transfection mixture for 4 h at 37 °C in an atmosphere of 10% CO2. The cells were then incubated for ∼40–48 h in fresh DMEM containing 5% charcoal stripped FBS with or without various concentrations of ligands. Cell lysates were produced using reporter lysis buffer according to the manufacturer's directions. Luciferase activity in cell extracts was determined using luciferase assay buffer in an ML3000 luminometer. β-Galactosidase activity was determined using β-d-galactopyranoside as described previously. Luciferase activities were normalized to β-galactosidase activities individually for each well. When assayed for antagonist activity of Lithocholic acid/LCA, cells were incubated with increasing concentrations of LCA in the presence of 100 nm GW4064.

Animal/Disease Models: Male mice (C57BL/6)[4].
Doses: 0.6% LCA-supplement diet, with the AIN93G diet as a control
Route of Administration: in diet, for 6 days
Experimental Results: Induced liver injury. Activated TGFβ-SMAD3 signaling. Increased serum ALP activities.

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Animal/Disease Models: Male mice (C57BL/6)[2].
Doses: 125 mg/kg, dissolved in corn oil
Route of Administration: ip, twice a day for four days
Experimental Results: Induced liver injury, generated necrosis and neutrophilic -granulocytic infiltrate (H&E staining). Increased AST, ALT and ALP level.

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Animals studies [2]
Male C57BL/6 mice (6–8 weeks old) were housed under temperature and humidity-controlled conditions with a 12 h light/12 h dark cycle. Thirty mice were randomly divided into six groups: (1) control of ANIT (ANIT-C); (2) ANIT; (3) control of DDC (DDC-C); (4) DDC; (5) control of Lithocholic acid/LCA (LCA-C); (6) LCA. ANIT dissolved in corn oil was given at a single dose of 75 mg/kg by gavage and mice were sacrificed 48 h after treatment (Fang et al., 2017; Tang et al., 2016). The administration of DDC was slightly modified on the previous reports (Dai et al., 2017; Fickert et al., 2007). DDC dissolved in corn oil was given at an oral dose 100 mg/kg for seven consecutive days, and mice were euthanized 24 h after the last dose of DDC. LCA dissolved in corn oil was intraperitoneal administered at dose of 125 mg/kg twice a day for four days consecutively (Beilke et al., 2009; Owen et al., 2010) and sacrificed 24 h after the last dose of LCA. The mice treated with corn oil were used as the control groups. All the mice were sacrificed by CO2 inhalation. Subsequently, mice plasma and liver samples were collected for biochemical assay, histopathology and metabolomic analysis. Biochemical assay included ALT, AST and ALP. Part of liver tissue were stained with hematoxylin and eosin (H&E) for histopathology using the method described previously (Hu et al., 2018) and the others were stored at −80 °C.

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Animals and diets [4]
Male mice (C57BL/6), MAD homolog 3 (Smad3)-null mice, and background-matched wild-type mice were housed in temperature- and light-controlled rooms and given water and pelleted NIH-31 chow ad libitum. For the Lithocholic acid/LCA studies, mice were given 0.6% LCA-supplement diet with the AIN93G diet as a control. Three wild-type and three Smad3-null mice were fed the control diet, and five wild-type and four Smad3-null mice were given the LCA diet.

Metabolism / Metabolites
Lithocholic acid (24)C(14) is converted to 3α-6β-dihydroxy-5β-cholic acid in rat liver homogenate, undergoing 7σ hydroxylation, which confirms its hydroxylation with taurine to form a 3-sulfate ester. When labeled lithocholic acid salts were injected into patients with gallstones and healthy volunteers, most of the radioactivity in the bile (50-60%) was present as sulfate conjugates. The glycine conjugates showed a higher degree of sulfation than the taurine conjugates, indicating that the glycine conjugates are more readily sulfated. Known metabolites of lithocholic acid include 6α-hydroxylithocholic acid.

Interactions
The inhibitory effect on skin tumors decreases in the following order of acids: chenodeoxycholic acid, lithocholic acid, deoxycholic acid, and cholic acid. 16α-Cyanogenolone (5 mg, intraperitoneal injection, twice daily for 2 days) increased the 6β- and 7α-hydroxylation of lithocholic acid in rat liver microsomes by 2-fold and 3-4-fold, respectively, in vitro. This may be the reason for its prevention of lithocholic acid-induced gallstones. Lithocholic acid (24)C(14) is converted to 3α-6β-dihydroxy-5β-cholic acid in rat liver homogenate. Adding ethanol to the enzyme system inhibits the production of 3α,6β-dihydroxy-5β-cholic acid. Sodium lithiumcholate increases the incidence of colon tumors induced by MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) in both sterile and conventional rats (F344). /Sodium Lithium Cholate/
LCA was also tested as a promoter of N-nitrobis(2-hydroxypropyl)amine (BHP)-induced carcinogenesis. Two groups of 5- to 6-week-old hamsters (number not specified) were subcutaneously injected with 500 mg/kg BHP weekly for 5 weeks. Group 3 received no further treatment. Group 4 received 0.5% LCA in their diet for 30 weeks. All animals underwent necropsy at week 35. There were no differences in feed intake or body weight between the two groups. There were also no differences in the number of liver lesions (Group 3: 15/15 hyperplastic nodules, 2/15 hepatocellular carcinomas, 1/15 cholangiocarcinomas; Group 4: 22/22 hyperplastic nodules, 3/22 hepatocellular carcinomas, 3/22 cholangiocarcinomas). However, there was a significant difference in the number of pancreatic tumors: Group 3 had 4/15 visible tumors, 5/15 carcinomas, and 4/15 adenomas; while Group 4 had 13/22 visible tumors, 15/22 carcinomas (P<0.04), and 2/22 adenomas. Under the experimental conditions, LCA alone was not carcinogenic, but it effectively promoted the development of BHP pancreatic cancer.

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Oral LD50 in mice: 3900 mg/kg. (NIH-NCI-EC-72-3252 Contract Progress Report, submitted by Litton Bionetics to the National Cancer Institute, NCI-EC-72-3252(1973))

[1]. Effect on blood lipids of very high intakes of fiber in diets low in saturated fat and cholesterol. N Engl J Med, 1993. 329(1): p. 21-6.

[2]. Metabolomic analysis of cholestatic liver damage in mice. Food Chem Toxicol. 2018 Jul 14;120:253-260.

[3]. Lithocholic bile acid selectively kills neuroblastoma cells, while sparing normal neuronal cells. Oncotarget, 2011. 2(10): p. 761-82.

[4]. TGF-beta-SMAD3 signaling mediates hepatic bile acid and phospholipid metabolism following lithocholic acid-induced liver injury. J Lipid Res, 2012. 53(12): p. 2698-707.

[5]. Lithocholic acid decreases expression of bile salt export pump through farnesoid X receptor antagonist activity. J Biol Chem. 2002 Aug 30;277(35):31441-7.

Hexagonal plate-like crystals (precipitated from alcohols) or prismatic crystals (precipitated from acetic acid) or white powder. (NTP, 1992)
Lycholic acid is a monohydroxy-5β-cholanic acid with an α-hydroxy substituent at the 3-position. It is a bile acid derived from chenodeoxycholic acid through bacterial conversion. It is a metabolite in both humans and mice and has anti-aging effects. It is a bile acid, monohydroxy-5β-cholanic acid, and C24 steroid. It is a conjugated acid of lithochondria.
Data on lithochlic acid in Homo sapiens has been reported.
A bile acid formed from chenodeoxycholic acid through bacterial action, usually conjugated with glycine or taurine. It acts as a surfactant, dissolving fats to promote absorption, and is also absorbed itself. It is used as a choleretic agent and choleretic drug. As mentioned above, bile duct obstruction is a pathological feature of primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). In three chemically induced cholestasis models, including TCDCA, T-α/β-MCA, and TH/UDCA, levels of various bile acids are generally elevated. In addition to elevated bile acid levels in these three cholestasis models, arginine levels are also decreased. The ANIT and DDC models disrupt lipid metabolism, leading to elevated levels of lysophosphatidylcholine (LPC) and carnitine in plasma and liver. Unlike the ANIT and LCA models, DCC-induced liver injury results in decreased hepatic glutathione (GSH) levels (Figure S3). The DDC model induces the accumulation of protoporphyrin IX (PPIX) in the liver, while PPIX levels are decreased in the ANIT and LCA models. Considering that PPIX is converted to heme for oxygen transfer in vivo, the decreased PPIX levels in the ANIT and LCA models may be an adaptive response to improved cell damage. In summary, these changes may partially reveal the differences among the three types of cholestatic liver injury and help to understand the underlying mechanisms of different types of cholestasis. The data in this paper confirm the advantages of metabolomics in the discovery of biomarkers for cholestatic liver injury. [2] Aging is one of the major risk factors for cancer. Anti-aging interventions such as drugs and diets can delay the onset of cancer. We recently found that lithocholic acid (LCA) can prolong life in a yeast cell aging model. This paper shows that LCA can kill neuroblastoma (NB) cell lines BE(2)-m17, SK-n-SH, SK-n-MCIXC and Lan-1 at concentrations that are non-cytotoxic to primary cultures of human neurons. In BE(2)-m17, SK-n-SH and SK-n-MCIXC cells, the antitumor effect of LCA is due to apoptosis. In contrast, LCA-induced cell death in Lan-1 cells is not caused by apoptosis. Low concentrations of LCA made BE(2)-m17 and SK-n-MCIXC cells more sensitive to hydrogen peroxide-induced mitochondrial-mediated apoptosis, but these concentrations of LCA made primary cultured human neurons resistant to this mode of cell death. LCA killed BE(2)-m17 and SK-n-MCIXC cell lines not only by activating the intrinsic (mitochondrial) apoptosis pathway driven by activation of mitochondrial outer membrane permeability and initiation caspase-9 activation, but also by activating the extrinsic (death receptor) apoptosis pathway mediated by initiation caspase-8. Based on these data, we proposed a mechanism by which LCA exerts a potent and selective antitumor effect in cultured human neuroblastoma (NB) cells. In addition, we found that LCA could kill cultured human breast cancer cells and mouse glioma cells, indicating that it has a broad antitumor effect on cancer cells derived from different tissues and organisms. [3] Bile acid export pump (BSEP) is the main bile acid transporter in the liver. Mutations in the BSEP gene lead to progressive intrahepatic cholestasis, a serious liver disease that impairs bile flow and causes irreversible liver damage. BSEP is a target for drug and aberrant bile acid metabolite inhibition and downregulation, which can lead to bile acid retention and intrahepatic cholestasis. In this study, we quantitatively analyzed the regulatory effects of FXR ligands on BSEP expression in primary human hepatocytes and HepG2 cells. We demonstrated that ligands of the nuclear receptor farnesol X (FXR) significantly regulate BSEP expression. Both the endogenous FXR agonist chenodeoxycholic acid (CDCA) and the synthetic FXR ligand GW4064 effectively increased BSEP mRNA expression in both cell types. This upregulation could be detected as early as 3 hours, and the ligand's regulatory potency towards BSEP was correlated with its intrinsic activity towards FXR. These results indicate that BSEP is a direct target of FXR and support recent reports that the BSEP promoter can be transactivated by FXR. In contrast to CDCA and GW4064, the hydrophobic bile acid lithocholic acid (LCA) is a potent inducing of cholestasis, significantly reducing BSEP expression. Previous studies have not identified LCA as a cellular FXR antagonist ligand, but we demonstrate here that LCA is a cellular FXR antagonist with partial agonist activity. In in vitro coactivator binding assays, LCA reduced CDCA- and GW4064-induced FXR activation with an IC50 value of 1 μM. In HepG2 cells, LCA also effectively antagonized GW4064-enhanced FXR transcriptional activation. These data suggest that the toxicity and cholestatic effects of LCA in animals may be due to its downregulation of BSEP via FXR. In summary, these observations indicate that FXR plays an important role in BSEP gene expression, and FXR ligands may be potential drugs for treating intrahepatic cholestasis. [5]

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LCA was recently identified as a PXR/SXR agonist ligand. PXR is considered the second bile acid receptor and plays a key role in liver detoxification (27, 28). However, there is currently no evidence that PXR is involved in the regulation of the BSEP gene. In fact, the PXR-specific ligand rifampin does not regulate BSEP expression, which suggests that PXR is not involved in the regulation of BSEP and further supports the conclusion that LCA downregulates BSEP expression by antagonizing FXR activity. BSEP expression is crucial for liver protection. As an important regulator of BSEP, FXR not only provides a molecular mechanism for FXR-mediated BSEP gene regulation, but also suggests that FXR ligands may be potential drugs for the treatment of intrahepatic cholestasis and lipid metabolism disorders. [5]

C24H40O3

376.57

376.297

C, 76.55; H, 10.71; O, 12.75

434-13-9

Allolithocholic acid;2276-94-0;Isoallolithocholic acid;2276-93-9;Isolithocholic acid;1534-35-6;Lithocholic acid-d4;83701-16-0;Lithocholic acid-d5;52840-06-9

9903

White to off-white solid powder

1.1±0.1 g/cm3

511.0±23.0 °C at 760 mmHg

183-188 °C(lit.)

276.9±19.1 °C

0.0±3.0 mmHg at 25°C

1.528

6.7

2

3

4

27

574

9

C[C@H](CCC(=O)O)[C@H]1CC[C@@H]2[C@@]1(CC[C@H]3[C@H]2CC[C@H]4[C@@]3(CC[C@H](C4)O)C)C

SMEROWZSTRWXGI-HVATVPOCSA-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

3alpha-Hydroxy-5beta-cholan-24-oic acid

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.52 mM) 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 20.8 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: ≥ 2.08 mg/mL (5.52 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: ≥ 1 mg/mL (2.66 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% 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 10.0 mg/mL clear EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 4: ≥ 1 mg/mL (2.66 mM) (saturation unknown) in 10% EtOH + 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 10.0 mg/mL clear EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix well.
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 5: ≥ 1 mg/mL (2.66 mM) (saturation unknown) in 10% EtOH + 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 10.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well.

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