Microbial Metabolite; isomer of Lithocholic acid; The targets of Isolithocholic acid involve multiple cellular signaling pathways. Studies have demonstrated its ability to inhibit multiple key steps of C. difficile spore germination and growth. Furthermore, as an agonist of GPBAR1 (G protein-coupled bile acid receptor 1), this compound may regulate intestinal L-cell differentiation and GLP-1 secretion through this receptor. Notably, its stereoisomer Isoallolithocholic acid has been shown to regulate T cell differentiation by modulating RORγt and FoxP3 expression, and although this activity has not been explicitly reported for Isolithocholic acid, the immunomodulatory functions of the bile acid family suggest it may participate in similar metabolic regulatory networks.

In cell-free and cellular systems, Isolithocholic acid exhibits multiple biological activities. It demonstrates significant inhibitory effects against C. difficile: at 0.01% concentration, it does not inhibit spore germination of CF5 and M120 strains, but at higher concentrations (0.1%) shows significant inhibition; at 0.00003%, it inhibits the growth of strains CD196, M68, CF5, 630, and BI9, and significantly reduces toxin activity of strains CF5, BI9, M120, and 630; at 0.0003%, toxin activity is significantly reduced in all tested strains except R20291 and M120. Furthermore, its stereoisomer Isoallolithocholic acid reduces Th17 cell differentiation by approximately 50% at 20 μM, independent of RORγt expression. This compound also enhances FoxP3 expression and promotes regulatory T cell (Tregs) differentiation by inducing mitochondrial reactive oxygen species (mitoROS) production.

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At a concentration of 0.01 percent, isolitocholic acid does not significantly impede CF5 and M120 spore germination and outgrowth, but at a higher concentration of 0.1%, it does. Isolithocholic acid (0.00003%) significantly lowers the toxin activity of strains CF5, BI9, M120, and 630 and inhibits the growth of CD196, M68, CF5, and BI9. With the exception of R20291 and M120, isolithocholic acid (0.0003%) causes all strains to exhibit a considerable drop in toxin activity[3].

In vivo activity of Isolithocholic acid is primarily studied using animal models. In a high-fat diet (HFD)-induced obesity rat model, fecal Isolithocholic acid levels in the HFD group were significantly decreased from day 28 compared to rats fed a normal diet, suggesting that obesity may affect the metabolism or excretion of this bile acid. Its stereoisomer Isoallolithocholic acid has shown immunomodulatory activity in vivo: in B6 mice, dietary administration of 0.03% Isoallolithocholic acid for 7 days enhanced the regulatory T cell (Tregs) population. GPBAR1 agonists (such as lithocholic acid) selectively increase L-cell density and enhance GLP-1 secretory capacity in vivo, improving glucose tolerance, suggesting that Isolithocholic acid may have similar metabolic regulatory functions.

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In comparison to rats on a regular diet, the high fat diet (HFD) group's fecal Isolithocholic acid levels clearly decreased starting on day 28[4].

Binding of Isolithocholic acid to the GPBAR1 receptor can be assessed using cell-based functional assays. A typical protocol: GPBAR1-expressing cell lines are seeded in 96-well plates and incubated with varying concentrations of Isolithocholic acid (0.1-100 μM). Receptor activation is quantified by measuring intracellular cAMP accumulation using homogeneous time-resolved fluorescence (HTRF) or enzyme-linked immunosorbent assay (ELISA) methods. For C. difficile spore germination inhibition assays: C. difficile spores are suspended in buffer containing taurocholic acid (as a germination inducer) with different concentrations of Isolithocholic acid (0.00003%-0.1%), incubated under anaerobic conditions at 37°C, and spore germination rates are assessed by microscopy counting or OD600 absorbance changes.

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Researchers have examined the mechanism for the bacterial transformation of chenodeoxycholic acid and lithocholic acid into the corresponding 3 beta-hydroxy epimers with the use of 3 alpha- and 3 beta-tritiated bile acids. The 3-oxo bile acids were transformed into the 3 alpha- (85%) and 3 beta- (15%) hydroxy bile acids after 20-hr incubation with Clostridium perfringens. Approximately 75% radioactivity was recovered in the aqueous medium when [3 beta-3H]chenodeoxycholic acid or [3 beta-3H]lithocholic acid was incubated with the bacteria, and approximately 15% of radioactivity in the bile acid fraction was associated with the 3 alpha-position of the iso-bile acids. When [3 beta-3H]chenodeoxycholic acid was incubated with unlabeled 3-oxo-5 beta-cholanoic acid, tritiated litho- and iso-lithocholic acids were recovered. These results can be explained only when a 3-oxo intermediate is postulated, and the 3 beta-hydrogen in the bile acids is transferred by the bacterial coenzyme (NAD+ or NADP+) to the 3 alpha-position in the iso-bile acids during the reduction of the 3-oxo compounds [1].

Isolithocholic acid is commonly used in cellular assays to study its effects on C. difficile or immunomodulatory functions. A typical protocol (C. difficile growth inhibition assay): C. difficile strains (e.g., CD196, M68, BI9, 630) are inoculated in BHI medium with varying concentrations of Isolithocholic acid (0.00003%-0.1%) and incubated under anaerobic conditions at 37°C for 24-48 hours. Bacterial growth density is measured by OD600, and toxin activity in the supernatant is assessed using Vero cell cytotoxicity assays. For T cell differentiation studies (based on stereoisomer protocols): CD4+ T cells are isolated from mouse spleens and cultured under Th17-polarizing conditions (IL-6 and TGF-β) with 20 μM Isolithocholic acid (or its isomer). After 72 hours of culture, Th17 cell proportions are assessed by detecting IL-17A expression via flow cytometry, or Treg differentiation is detected by FoxP3 staining.

In vivo studies of Isolithocholic acid typically employ metabolic disease animal models. A typical protocol (high-fat diet-induced obesity rat model): Male Sprague-Dawley rats are randomly divided into normal diet group and high-fat diet (HFD) group. From day 0 to day 56, fecal samples are collected at regular intervals (e.g., days 0, 7, 14, 21, 28, 35, 42, 49, 56), and Isolithocholic acid and other bile acid levels in feces are detected by LC-MS/MS. At the endpoint of day 56, serum, liver, and intestinal tissues are collected for further analysis. For immunomodulatory studies (based on stereoisomer protocols): B6 mice are randomly grouped and administered 0.03% Isolithocholic acid (or its isomer) via diet for 7 days. In some experiments, anti-CD3 antibody treatment may be combined to activate T cells. Spleens and mesenteric lymph nodes are collected, and CD4+ FoxP3+ Treg cell proportions are detected by flow cytometry.

As a member of the bile acid family, the pharmacokinetic behavior of Isolithocholic acid follows the enterohepatic circulation pattern of bile acids. Predicted physicochemical properties: LogP is approximately 6.7, indicating high lipophilicity; hydrogen bond donor count is 2, hydrogen bond acceptor count is 3, and rotatable bond count is 4. This compound exists primarily as an anion at physiological pH and requires specific carrier proteins for transmembrane transport. It is produced by gut microbiota in the colon, reabsorbed into the liver via the portal vein, subsequently secreted into bile, and then enters the intestine, forming an enterohepatic circuit. Under obese conditions, fecal Isolithocholic acid levels are significantly reduced, suggesting that metabolic status may affect its in vivo levels. Storage conditions: The powder is stable for 3 years at -20°C and 2 years at 4°C; once dissolved, it can be stored for 6 months at -80°C and 1 month at -20°C. Solubility in DMSO is 20 mg/mL.

According to the available Material Safety Data Sheet (MSDS), Isolithocholic acid is classified as a non-hazardous substance or mixture and is considered safe under normal laboratory handling conditions. This product is for research use only and not for human or veterinary use. As an endogenous bile acid, it maintains homeostasis at normal physiological concentrations through enterohepatic circulation, but high concentrations of bile acids exhibit potential toxicity, primarily manifesting as disruptive effects on cell membranes. IARC, ACGIH, NTP, and OSHA do not classify this compound or its components as human carcinogens. It should be handled by technically qualified persons following standard laboratory practices, and wearing personal protective equipment is recommended. It should be noted that bile acids structurally similar to Isolithocholic acid may exhibit cytotoxicity to certain cell types at high concentrations, and their abnormal accumulation under conditions of metabolic dysregulation may contribute to pathological processes.

[1]. Transformation of bile acids into iso-bile acids by Clostridium perfringens: possible transport of 3 beta-hydrogen via the coenzyme. Hepatology. 1985;5(6):1126-1131.

[2]. The metabolism of lithocholic acid and lithocholic acid-3-alpha-sulfate by human fecal bacteria. Lipids. 1982;17(7):477-482.

[3]. Inhibition of spore germination, growth, and toxin activity of clinically relevant C. difficile strains by gut microbiota derived secondary bile acids. Anaerobe. 2017;45:86-100.

[4]. Alterations of Bile Acids and Gut Microbiota in Obesity Induced by High Fat Diet in Rat Model. J Agric Food Chem. 2019;67(13):3624-3632.

Isolithocholic acid is a monohydroxy-5β-cholanic acid with a β-hydroxy substituent at the 3-position. It is the 3β-hydroxy epimer of lithocholic acid. It is a metabolite in humans, rats, and exogenous substances. It is a bile acid, a 3β-hydroxy steroid, a monohydroxy-5β-cholanic acid, and a C24 steroid. It is a conjugated acid of isolithocholic acid. Isolithocholic acid is a bile acid formed from chenodeoxycholic acid by bacterial action, usually conjugated with glycine or taurine. It acts as a detergent, dissolving fats to promote absorption, and is itself absorbed. It is used as a choleretic agent and choleretic drug. Changes in the epidemiology of Clostridium difficile infection over the past few decades have presented significant challenges to the treatment of Clostridium difficile-related diseases. Gastrointestinal flora can resist Clostridium difficile colonization, and increasing evidence suggests that secondary bile acids (SBAs) derived from gut microbiota play a crucial role in this process. We hypothesized that Clostridium difficile strains of different ages and ribosomal types exhibit varying susceptibility to taurocholic acid (TCA) mediated spore germination and growth, growth, and toxin production. This study used Clostridium difficile strains R20291 and CD196 (ribosomal type 027), M68 and CF5 (017), 630 (012), BI9 (001), and M120 (078) to investigate TCA-mediated spore germination and growth, growth, and toxin activity. We also examined the presence and absence of bile acids from gut microbiota (deoxycholic acid, isodeoxycholic acid, lithocholic acid, isolithocholic acid, ursodeoxycholic acid, ω-mouse cholic acid, and porcine deoxycholic acid) in the large intestine of humans and mice. The results showed that germination rates, growth kinetics, and toxin activities differed among different Clostridium difficile strains in the absence of added bile acids. Among them, Clostridium difficile strain M120 differed significantly from other strains, exhibiting strong germination and growth capabilities, but significantly lower toxic activity than other strains. Many bile acids (SBAs) can inhibit tricarboxylic acid cycle (TCA)-mediated spore germination and growth, as well as toxic activity, in a dose-dependent manner, but the degree of inhibition and drug resistance vary among different strains and ribosomal subtypes. This study suggests that clinically relevant Clostridium difficile strains may produce different responses when exposed to bile acids present in the gastrointestinal tract. [3]

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Obesity has become a global health problem and has attracted widespread public attention. This study aimed to elucidate the role of bile acids and their interaction with gut microbiota in the development of obesity using a high-fat diet (HFD) induced rat obesity model. We collected fecal, plasma, liver, and intestinal tissue samples and developed a method for quantifying bile acids using an ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) strategy. Subsequently, we evaluated the bile acid flux in the collected biomatrix. We found that regardless of dietary regimen, taurine-conjugated bile acids were the predominant bile acids in the liver, while unconjugated bile acids were mainly found in plasma. However, a high-fat diet (HFD) led to a slight increase in the total bile acid pool, particularly a significant increase in plasma and liver tissue levels of deoxycholic acid (DCA) (control group: 138.67 ± 37.225 nmol/L; HFD group: 242.61 ± 43.16 nmol/L, p = 0.014) and taurine-deoxycholic acid (TDCA) (control group: 2.8 ± 0.247 nmol/g; HFD group: 4.5 ± 0.386 nmol/g, p = 0.0018), consistent with increased DCA levels in intestinal tissue and feces. These changes were associated with increased abundance in genera such as Blautia, Coprococcus, Intestinimonas, Lactococcus, Roseburia, and Ruminococcus. Our study revealed the flux of bile acids during obesity development and their relationship with the gut microbiota, and elucidated the adverse health effects of a high-fat diet. [4]

C24H40O3

376.57

376.297

C, 76.55; H, 10.71; O, 12.75

1534-35-6

Lithocholic acid;434-13-9;Isoallolithocholic acid;2276-93-9

164853

White to off-white solid powder

1.1±0.1 g/cm3

511.0±23.0 °C at 760 mmHg

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-WFVDQZAMSA-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

(4R)-4-[(3S,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]pentanoic acid

Isolithocholic acid; 1534-35-6; beta-Lithocholic acid; 3-Epilithocholic acid; beta-Lithocholanic acid; (4R)-4-[(3S,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]pentanoic acid; 3beta-Hydroxy-5beta-cholan-24-oic Acid; Iso-LCA;

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: 28.57 mg/mL (75.87 mM)

Solubility in Formulation 1: ≥ 2.86 mg/mL (7.59 mM) (saturation unknown) in 10% DMSO + 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 28.6 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL 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 2: ≥ 2.86 mg/mL (7.59 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 28.6 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 3: ≥ 2.86 mg/mL (7.59 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 28.6 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.)