Human Endogenous Metabolite; The targets of Taurolithocholic acid involve multiple signaling pathways and receptors. It is an agonist of the G protein-coupled bile acid receptor TGR5, exerting anti-inflammatory effects by activating TGR5 and downregulating the NF-κB signaling pathway. In mouse hypothalamic GT1-7 cells, TLCA activates both TGR5 and FXR receptors, subsequently upregulating STAT3 phosphorylation and SOCS3 expression. This compound is a potent inhibitor of UDP-glucuronosyltransferase (UGT) enzymes, particularly the UGT1A4 isoform, with inhibitory potency stronger than other bile acids. Additionally, TLCA acts as a Ca²⁺ agonist.

In cell-free and cellular systems, Taurolithocholic acid exhibits multiple biological activities. As a UGT enzyme inhibitor, TLCA shows the strongest inhibition against UGT1A4 (lowest Ki value), with inhibitory potency in the order: UGT1A4 > UGT2B7 > UGT1A3 > UGT1A1. In RAW264.7 macrophages, TLCA downregulates the NF-κB signaling pathway via the TGR5 receptor, significantly reducing LPS and IFN-γ-induced inflammatory responses. In mouse hypothalamic GT1-7 cells, treatment with 10 μmol/L TLCA for 24 hours significantly increases POMC mRNA expression and the production of α-MSH (an anorexigenic neuropeptide), involving p-STAT3, p-AKT, and SOCS3 signaling proteins. Additionally, TLCA exhibits selective inhibitory activity against HIV-1 replication.

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At low doses (5 µM), tTaurolithocholic acid (TLCA) tends to raise the membrane-associated fraction of the e-isoform of PKC by 44.1% ± 40.2%[1]. The activation of mobile PKC isoforms requires the selective translocation of PKC's epsilon-isoform to hepatocellular membranes, which is induced by TLCA (10 µM)[1].

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The protein kinase C (PKC) family of isoenzymes plays a key role in the regulation of hepatocellular secretion. The hydrophobic and cholestatic bile acid, Taurolithocholic acid (TLCA), acts as a potent Ca++ agonist in isolated hepatocytes. However, its effect on PKC isoforms has not been elucidated. Here we investigate the effects of TLCA at low micromolar concentrations on the distribution of PKC isoforms and on membrane-associated PKC activity. The distribution of PKC isoforms was determined in isolated rat hepatocytes in short-term culture using Western blotting and immunofluorescence techniques. PKC activity was measured radiochemically. TLCA (10 micromol/L) induced selective translocation of epsilon-PKC by 47.9% +/- 20.5% (P <.02 vs. controls; n = 7), but not of alpha-, delta-, and zeta-PKC to the hepatocellular membranes, whereas the phorbol ester, phorbol 12-myristate 13-acetate (PMA) (1 micromol/L) caused translocation of all mobile isoforms, alpha-, delta-, and epsilon-PKC, as shown by immunoblotting. Immunofluorescence studies demonstrated selective translocation of epsilon-PKC to the canalicular membranes of isolated rat hepatocyte couplets by TLCA (10 micromol/L), but predominant translocation to intracellular and basolateral membranes by PMA (1 micromol/L). Both TLCA (10 micromol/L) and PMA (1 micromol/L) stimulated membrane-bound PKC activity by 60.5% +/- 45. 8% (P <.05 vs. controls; n = 5) and 72.4% +/- 37.2% (P <.05; n = 5), respectively. TLCA at lower concentrations (5 micromol/L) was less effective. Because activation of epsilon-PKC has been associated with impairment of vesicle-mediated targeting and insertion of membrane proteins in secretory cells, it is attractive to speculate that TLCA reduces bile secretory capacity of the liver cell by activation of epsilon-PKC at the canalicular membrane.[1]

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PI3K-dependent PKB (PKB/Akt) Activity in Isolated Rat Hepatocytes [2]
The amount of phospho-PKB(Ser-473), a sensitive read-out of the activation of the PI3K pathway (27, 32), was markedly enhanced by Taurolithocholic acid /TLCA (5 μmol/liter) in hepatocytes in short term culture (Fig. 7) and reached levels up to 194 ± 46% of controls after 60 min (p < 0.005versus control; p < 0.05 versusTUDCA; p < 0.01 versus TCA). In contrast, TUDCA (10 μmol/liter) only transiently increased PKB activity, whereas TCA (10 μmol/liter) had no effect under the experimental conditions chosen (Fig. 7). Thus, TLCA markedly affected PI3K activity in isolated hepatocytes in vitro, whereas TUDCA exerted only minor transient effects on the PI3K pathway when administered at low micromolar concentrations.

In vivo activity of Taurolithocholic acid is primarily studied through its cholestatic effects. In rat models, intravenous administration of TLCA (3 μmol/100 g body weight) inhibits canalicular excretion of taurocholate by 58% and increases plasma reflux by 96%. This compound impairs bile salt secretion by inducing internalization of the canalicular bile salt transporter Bsep into a cytosolic vesicular compartment without affecting F-actin cytoskeletal organization. Pretreatment with dibutyryl-cAMP reverses TLCA-induced Bsep internalization and cholestasis. In rats, TLCA pretreatment (5 μmol/100 g body weight) increases total clearance of acetaminophen by 20%, as well as partial clearances for sulfation (12%) and glucuronidation (85%).

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Taurolithocholic acid (TLCA) in rat hepatocyte couplets and perfused rat livers exhibits cholestatic effects through PI3K-dependent mechanisms[2].
Taurolithocholic acid (TLCA) is a potent cholestatic agent. Our recent work suggested that TLCA impairs hepatobiliary exocytosis, insertion of transport proteins into apical hepatocyte membranes, and bile flow by protein kinase Cepsilon (PKCepsilon)-dependent mechanisms. Products of phosphatidylinositol 3-kinases (PI3K) stimulate PKCepsilon. We studied the role of PI3K for TLCA-induced cholestasis in isolated perfused rat liver (IPRL) and isolated rat hepatocyte couplets (IRHC). In IPRL, TLCA (10 micromol/liter) impaired bile flow by 51%, biliary secretion of horseradish peroxidase, a marker of vesicular exocytosis, by 46%, and the Mrp2 substrate, 2,4-dinitrophenyl-S-glutathione, by 95% and stimulated PI3K-dependent protein kinase B, a marker of PI3K activity, by 154% and PKCepsilon membrane binding by 23%. In IRHC, TLCA (2.5 micromol/liter) impaired canalicular secretion of the fluorescent bile acid, cholylglycylamido fluorescein, by 50%. The selective PI3K inhibitor, wortmannin (100 nmol/liter), and the anticholestatic bile acid tauroursodeoxycholic acid (TUDCA, 25 micromol/liter) independently and additively reversed the effects of TLCA on bile flow, exocytosis, organic anion secretion, PI3K-dependent protein kinase B activity, and PKCepsilon membrane binding in IPRL. Wortmannin also reversed impaired bile acid secretion in IRHC. These data strongly suggest that TLCA exerts cholestatic effects by PI3K- and PKCepsilon-dependent mechanisms that are reversed by tauroursodeoxycholic acid in a PI3K-independent way [2].

Interaction of Taurolithocholic acid with UGT enzymes can be assessed using in vitro inhibition kinetic assays. A typical protocol: Human liver microsomes (or recombinant single-isoform UGT enzymes) are preincubated with varying concentrations of TLCA (0-200 μM) in UGT reaction buffer (e.g., Tris-HCl, pH 7.4), alamethicin is added to disrupt microsomal membranes, and reactions are initiated by adding substrates and UDPGA. Glucuronide conjugate formation is detected by HPLC or LC-MS/MS to calculate IC₅₀ and Ki values. For TGR5 receptor binding assays, cell-based cAMP assays can be used to evaluate TLCA agonist activity by measuring intracellular cAMP accumulation following receptor activation.

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PKB/Akt activity in isolated rat hepatocytes was determined by an immunoblotting technique. In brief, 4 h after plating (see above) cells were incubated for 5, 15, 30, and 60 min with Taurolithocholic acid /TLCA (5 μmol/liter; at concentrations >5 μmol/liter, TLCA caused visible damage in isolated hepatocytes in short term culture), TUDCA (10 μmol/liter), TCA (10 μmol/liter), or the carrier Me2SO only (control, 0.1%, v/v). Culture dishes were then placed on ice, and cells were scraped and immediately shock-frozen (−80 °C). Shock-frozen cells were homogenized in ice-cold lysis buffer (1 ml/100 mg) and processed as described above [2].

Taurolithocholic acid is widely used in cellular assays to study inflammation regulation and bile acid signaling pathways. A typical protocol (anti-inflammatory study): RAW264.7 macrophages are seeded in culture plates, pretreated with varying concentrations of TLCA (e.g., 10, 50, 100 μM) for 1 hour, then stimulated with LPS and IFN-γ to induce inflammatory responses. Supernatants are collected to detect inflammatory cytokines (e.g., TNF-α, IL-6), and cell lysates are collected to detect NF-κB p65 phosphorylation levels and TGR5 expression by Western blotting. For neuropeptide expression studies: Mouse hypothalamic GT1-7 cells are treated with 10 μmol/L TLCA for 24 hours, POMC mRNA expression is detected by real-time PCR, α-MSH production is measured by ELISA, and p-STAT3, p-AKT, and SOCS3 protein levels are detected by Western blotting.

In vivo studies of Taurolithocholic acid primarily employ rat cholestasis models. A typical protocol: Male Sprague-Dawley rats (approximately 250-300 g) are anesthetized and undergo jugular vein and common bile duct cannulation. TLCA (3-5 μmol/100 g body weight, dissolved in saline or bovine serum albumin solution) is administered intravenously, bile samples are collected, and bile flow is measured every 5-10 minutes. Bile acid secretion in bile is detected by HPLC or radiolabeling methods. For Bsep localization studies, animals are euthanized, liver tissues are collected for frozen sectioning, and Bsep immunofluorescence staining is performed using specific antibodies, with subcellular localization changes observed by confocal microscopy. Dibutyryl-cAMP (10 μmol/100 g body weight) can be administered 15 minutes prior to assess its preventive effect against TLCA-induced cholestasis.

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Bile acid secretion by IRHC was assessed by measuring the hepatocellular uptake and secretion of 1 μmol/liter cholylglycylamido fluorescein (CGamF) into the canalicular space as previously described. CGamF was synthesized according to Schteingart et al. and was kindly provided by Dr. Alan Hofmann. Four hours after isolation, hepatocytes (on coverslips) were briefly transferred to HEPES buffer. Then, cells were pretreated for 15 min at 37 °C with (i) Me2SO (0.1%, v/v), (ii) 100 nmol/liter wortmannin and Me2SO, (iii) Me2SO for 5 min, and 2.5 μm Taurolithocholic acid /TLCA (in Me2SO, 0.1%, v/v) for 10 min, (iv) 100 nmol/liter wortmannin and Me2SO for 5 min, and 100 nmol/liter wortmannin and 2.5 μm Taurolithocholic acid /TLCA (in Me2SO, 0.1%, v/v) for 10 min, (v) Me2SO for 5 min and 5 μmol/liter Taurolithocholic acid /TLCA (in Me2SO, 0.1%, v/v) for 10 min, and (vi) 100 nmol/liter wortmannin and Me2SO for 5 min, and 100 nmol/liter wortmannin and 5 μmol/liter Taurolithocholic acid /TLCA for 10 min. Cells were then transferred for 5 min to HEPES buffer containing 1 μmol/liter fluorescent CGamF at 37 °C to allow adequate loading of the fluorescent bile acid and transferred back for 10 min to their previous dishes (i-vi). Hepatocyte secretion was stopped by placing coverslips in ice-cold HEPES buffer on ice, and cells were viewed immediately on a Zeiss LSM 510 microscope (Thornwood, NY). Laser settings were optimized for a dynamic range to avoid saturation of the fluorescence. The same settings were used for all conditions. Cells were analyzed on the confocal laser scanning microscope by one investigator (C. J. Soroka) who was blinded to the experimental conditions. Couplets were selected based upon the presence of a well defined canalicular space as determined under bright field optics. Images were then acquired with rapid scanning to avoid quenching of the fluorescence. Quantitation of uptake (uptake = (F° cell + F° can)/μm2) and secretion (% secretion = [F° can/(F° cell + F° can)] × 100) of CGamF was performed as previously published, except that NIH Image software was used.[2]

As a taurine-conjugated bile acid, the pharmacokinetic behavior of Taurolithocholic acid follows the enterohepatic circulation pattern of bile acids. Its molecular weight is 483.7 g/mol, and its melting point is 212-213°C. The predicted pKa is 1.42±0.50, and it is soluble in DMSO. This compound is primarily excreted via bile, and its levels are significantly elevated under cholestatic conditions. TLCA inhibits the activity of multiple UGT enzymes, which may interfere with the metabolism of both endogenous and exogenous compounds. Storage conditions: The powder should be kept dry and sealed at -20°C. This product is for research use only and not for human use.

Taurolithocholic acid is well-known for its potent cholestatic toxicity. In rat models, intravenous administration of TLCA (3-5 μmol/100 g body weight) dose-dependently induces cholestasis, reducing bile flow by 45%-85%. Its toxic mechanism involves internalization of the canalicular bile salt transporter Bsep, leading to impaired bile salt secretion. At the cellular level, TLCA induces hepatocyte apoptosis and inflammatory responses. However, it is worth noting that sulfated derivatives of TLCA (e.g., taurolithocholic acid 3-sulfate) exhibit anti-HIV-1 activity with lower cytotoxicity. According to GHS classification, the hazard symbols for TLCA include GHS02 and GHS08, with a signal word of "Danger". This compound is for research use only and not for human use.

[1]. Modulation of protein kinase C by taurolithocholic acid in isolated rat hepatocytes. Hepatology. 1999 Feb;29(2):477-82.

[2]. Taurolithocholic acid exerts cholestatic effects via phosphatidylinositol 3-kinase-dependent mechanisms in perfused rat livers and rat hepatocyte couplets. J Biol Chem. 2003 May 16;278(20):17810-8.

Taurolithocholic acid is a bile acid formed by the combination of lithocholic acid and taurine. It is a human metabolite. It is a monocarboxylic acid amide and a conjugate of bile acid and taurine. Functionally, it is related to lithocholic acid. It is a conjugated acid of Taurolithocholic acid salt. Taurolithocholic acid has been reported in Homo sapiens, bovine bacteria, and Aeromonas veronii, with relevant data. It is a bile salt formed by the combination of lithocholic acid and taurine in the liver, usually existing as a sodium salt. It can dissolve fats to promote absorption and can also be absorbed itself. It is a choleretic and a choleretic agent. Sodium taurollipate is a bile acid. Functionally, it is related to Taurolithocholic acid. See also: Sodium taurollipate (note moved to). In this study, the combined administration of PI3K inhibitors not only reversed TLCA-induced bile secretion disorders but also reversed cell damage reflected by lactate dehydrogenase release (Table I). The improvement in bile flow alone is insufficient to explain this effect, as while TUDCA also improved bile secretion in TLCA-treated livers, it failed to eliminate TLCA-induced cell damage in IPRL. Further research is needed to elucidate the role of PI3K in TLCA-induced acute hepatocellular injury. Current data suggest that PI3K may be a potential target for future anti-cholestasis therapies. However, it is noteworthy that PI3K may activate a survival pathway in rat hepatocytes treated with the hydrophobic bile acid taurine chenodeoxycholic acid (TCDCA), which protects hepatocytes from TCDCA-induced damage both in vitro and in vivo (Rust C, unpublished observation). Interestingly, the taurine chenodeoxycholic acid-induced survival pathway does not involve PKB activation in vitro. Therefore, different bile acids may have different effects on PI3K and PKB-mediated processes in hepatocytes. Whether the involvement of different PI3K isoforms or their effects in different subcellular compartments leads to these different effects of bile acids on PI3K and PKB remains to be elucidated. In summary, this study shows that TLCA-induced bile flow, hepatobiliary exocytosis, secretion of bile acids and other organic anions, and hepatocyte damage are mediated by a PI3K-dependent mechanism and a hypothesized PKCε-dependent mechanism. TUDCA reversed the inhibitory effect of TLCA on bile secretion through a PI3K-independent mechanism. [2]

C26H45NO5S

483.7

483.302

C, 64.56; H, 9.38; N, 2.90; O, 16.54; S, 6.63

516-90-5

6042-32-6; 516-90-5

439763

White to off-white solid at room temperature

1.169g/cm3

81-82ºC

1.54

6.347

3

5

7

33

825

9

CC(CCC(=O)NCCS(=O)(=O)O)C1CCC2C1(CCC3C2CCC4C3(CCC(C4)O)C)C

QBYUNVOYXHFVKC-GBURMNQMSA-N

InChI=1S/C26H45NO5S/c1-17(4-9-24(29)27-14-15-33(30,31)32)21-7-8-22-20-6-5-18-16-19(28)10-12-25(18,2)23(20)11-13-26(21,22)3/h17-23,28H,4-16H2,1-3H3,(H,27,29)(H,30,31,32)/t17-,18-,19-,20+,21-,22+,23+,25+,26-/m1/s1

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

Lithocholic acid; taurine conjugate; TAUROLITHOCHOLIC ACID; 516-90-5; 2-[[(4R)-4-[(3R,5R,8R,9S,10S,13R,14S,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethanesulfonic acid; CHEBI:36259; DTXSID20965925; N-(3alpha-hydroxy-5beta-cholan-24-oyl)-taurine; ST 24:1;O2;T; Acid, Taurolithocholic; Taurolithocholic acid; Lithocholyltaurine

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: 16.7 mg/mL (34.5 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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