Endogenous metabolite; glycine-conjugated secondary bile acid

Lithocholic acid (LCA) is a bile acid associated with adverse effects, including cholestasis, and it exists in vivo mainly as conjugates known as Glycolithocholic acid/glyco-LCA (GLCA) and tauro-LCA (TLCA). Tamoxifen has been linked to the development of cholestasis, and it inhibits sulfotransferase 2A1 (SULT2A1)-catalyzed dehydroepiandrosterone (DHEA) sulfonation. The present study was done to characterize the sulfonation of LCA, GLCA, and TLCA and to investigate whether triphenylethylene (clomifene, tamoxifen, toremifene, ospemifene, droloxifene), benzothiophene (raloxifene, arzoxifene), tetrahydronaphthalene (lasofoxifene, nafoxidine), indole (bazedoxifene), and benzopyran (acolbifene) classes of selective estrogen receptor modulator (SERM) inhibit LCA, GLCA, and TLCA sulfonation. Human recombinant SULT2A1, but not SULT2B1b or SULT1E1, catalyzed LCA, GLCA, and TLCA sulfonation, whereas each of these enzymes catalyzed DHEA sulfonation. LCA, GLCA, and TLCA sulfonation is catalyzed by human liver cytosol, and SULT2A1 followed the substrate inhibition model with comparable apparent K m values (≤1 µM). Each of the SERMs inhibited LCA, GLCA, and TLCA sulfonation with varying potency and mode of enzyme inhibition. The potency and extent of inhibition of LCA sulfonation were attenuated or increased by structural modifications to toremifene, bazedoxifene, and lasofoxifene. The inhibitory effect of raloxifene, bazedoxifene, and acolbifene on LCA sulfonation was also observed in HepG2 human hepatocellular carcinoma cells. Overall, among the SERMs investigated, bazedoxifene and raloxifene were the most effective inhibitors of LCA, GLCA, and TLCA sulfonation. These findings provide insight into the structural features of specific SERMs that contribute to their inhibition of SULT2A1-catalyzed LCA sulfonation. Inhibition of LCA, GLCA, and TLCA detoxification by a SERM may provide a biochemical basis for adverse effects associated with a SERM [3].

In the patients with heterozygous FH type IIa, we observed both a significant decrease in the molar percentages of glycochenodeoxycholic acid, glycoursodeoxycholic acid, and Glycolithocholic acid, and a significant increase of taurochenodeoxycholic acid compared to the corresponding values in the controls. Bile analysis of six patients was reexamined during probucol treatment after 16 weeks. Probucol significantly lowered serum cholesterol levels. However, biliary lipid composition and individual bile acid proportions was not altered by the treatment. The results suggest that most of the patients with heterozygous FH have supersaturated bile and are predisposed to cholesterol gallstone formation. In addition, the mechanism by which probucol lowers serum cholesterol appears to be independent of any change in the metabolism of biliary lipid.[1]

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Results: Thirty-two UC patients and twenty-three HCs were enrolled in this study. It was found that the diversity of gut microbiota in UC patients was reduced compared with that in HCs. Firmicutes, Clostridium IV, Butyricicoccus, Clostridium XlVa, Faecalibacterium, and Roseburia were significantly decreased in patients with UC (P = 3.75E-05, P = 8.28E-07, P = 0.0002, P = 0.003, P = 0.0003, and P = 0.0004, respectively). Proteobacteria, Escherichia, Enterococcus, Klebsiella, and Streptococcus were significantly enriched in the UC group (P = 2.99E-09, P = 3.63E-05, P = 8.59E-05, P = 0.003, and P = 0.016, respectively). The concentrations of fecal secondary BAs, such as lithocholic acid, deoxycholic acid, glycodeoxycholic acid, Glycolithocholic acid, and taurolithocholate, in UC patients were significantly lower than those in HCs (P = 8.1E-08, P = 1.2E-07, P = 3.5E-04, P = 1.9E-03, and P = 1.8E-02, respectively) and were positively correlated with Butyricicoccus, Roseburia, Clostridium IV, Faecalibacterium, and Clostridium XlVb (P < 0.01). The concentrations of primary BAs, such as taurocholic acid, cholic acid, taurochenodeoxycholate, and glycochenodeoxycholate, in UC patients were significantly higher than those in HCs (P = 5.3E-03, P = 4E-02, P = 0.042, and P = 0.045, respectively) and were positively related to Enterococcus, Klebsiella, Streptococcus, Lactobacillus, and pro-inflammatory cytokines (P < 0.01). The expression of TGR5 was significantly elevated in UC patients (0.019 ± 0.013 vs 0.006 ± 0.003, P = 0.0003). VDR expression in colonic mucosal specimens was significantly decreased in UC patients (0.011 ± 0.007 vs 0.016 ± 0.004, P = 0.033). Conclusion: Fecal BA profiles are closely related to the gut microbiota and serum inflammatory cytokines. Dysregulation of the gut microbiota and altered constitution of fecal BAs may participate in regulating inflammatory responses via the BA receptors TGR5 and VDR [2].

Optimization of the LCA, GLCA/Glycolithocholic acid, TLCA, and DHEA Sulfonation Assays [3]
LCA sulfonation assay in human liver cytosol was optimized previously (Bansal and Lau, 2016a). LCA and GLCA/Glycolithocholic acid sulfonation increased linearly up to 100 µg of cytosolic protein, whereas TLCA sulfonation increased linearly up to 80 µg of cytosolic protein (Supplemental Fig. S1, A–C). LCA, GLCA, and TLCA sulfonation catalyzed by recombinant SULT2A1 was linear up to 5 µg of enzyme (Supplemental Fig. S1, D–F). LCA sulfonation catalyzed by human liver cytosol was linear up to 45 minutes, whereas GLCA..

Background: Gut microbiota and its metabolites may be involved in the pathogenesis of inflammatory bowel disease. Several clinical studies have recently shown that patients with ulcerative colitis (UC) have altered profiles of fecal bile acids (BAs). It was observed that BA receptors Takeda G-protein-coupled receptor 5 (TGR5) and vitamin D receptor (VDR) participate in intestinal inflammatory responses by regulating NF-ĸB signaling. We hypothesized that altered profiles of fecal BAs might be correlated with gut microbiota and inflammatory responses in patients with UC. Aim: To investigate the changes in fecal BAs and analyze the relationship of BAs with gut microbiota and inflammation in patients with UC. Methods: The present study used 16S rDNA sequencing technology to detect the differences in the intestinal flora between UC patients and healthy controls (HCs). Fecal BAs were measured by targeted metabolomics approaches. Mucosal TGR5 and VDR expression was analyzed using immunohistochemistry, and serum inflammatory cytokine levels were detected by ELISA.[2]

[1]. Biliary lipid composition in heterozygous familial hypercholesterolemia and influence of treatment with probucol. Dig Dis Sci. 1994 Jul;39(7):1586-91.

[2]. Altered profiles of fecal bile acids correlate with gut microbiota and inflammatory responses in patients with ulcerative colitis. World J Gastroenterol. 2021 Jun 28;27(24):3609-3629.

[3]. Inhibition of Human Sulfotransferase 2A1-Catalyzed Sulfonation of Lithocholic Acid, Glycolithocholic Acid, and Taurolithocholic Acid by Selective Estrogen Receptor Modulators and Various Analogs and Metabolites. J Pharmacol Exp Ther. 2019 Jun;369(3):389-405.

[4]. Potent suppression of hydrophobic bile acids by aldafermin, an FGF19 analogue, across metabolic and cholestatic liver diseases. JHEP Rep. 2021 Feb 19;3(3):100255.

Glycolithocholic acid is a glycine conjugate of lithocholic acid. It is a bile acid glycine conjugate and also an N-acylglycine. It is functionally related to lithocholic acid. It is the conjugated acid of glycocholate. Glycolithocholic acid has been reported in both Homo sapiens and cattle, and relevant data are available. See also: Glycinecholate (note moved here). Background and Objectives: Elevated serum bile acid levels are associated with cirrhosis and an increased risk of liver-related diseases and death. This article reports a secondary analysis of the circulating bile acid profile of aldafermin, an engineered analog of the intestinal hormone fibroblast growth factor 19, in a prospective phase II study in patients with metabolic or cholestatic liver disease. Methods: 176 patients with biopsy-confirmed nonalcoholic steatohepatitis (NASH) accompanied by fibrosis and elevated liver fat content (proton density fat fraction ≥8% on magnetic resonance imaging) were treated for 12 weeks with 0.3 mg (n = 23), 1 mg (n = 49), 3 mg (n = 49), 6 mg (n = 28) adaphene or placebo (n = 27), respectively. 62 patients with primary sclerosing cholangitis (PSC) with elevated alkaline phosphatase (>1.5 times the upper limit of normal) were treated for 12 weeks with 1 mg (n = 21), 3 mg (n = 21) adaphene or placebo (n = 20), respectively. Serum samples were collected on day 1 and week 12 for bile acid protease and type III collagen neoepitope-specific N-terminal propeptide (Pro-C3), the latter being a direct indicator of fibrosis. Results: Aldafermin treatment significantly reduced serum bile acid levels in a dose-dependent manner. Notably, in patients with both non-alcoholic steatohepatitis (NASH) and primary sclerosing cholangitis (PSC), adafermin significantly reduced bile acids with high hydrophobic indices, such as deoxycholic acid, lithocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, and glycocholic acid. Furthermore, adafermin primarily inhibited glycine-conjugated bile acids, rather than taurine-conjugated bile acids. In patients with both NASH and PSC, changes in bile acid levels were associated with changes in the novel fibrosis marker Pro-C3. Pro-C3 detects novel epitopes in type III collagen formation. Conclusion: Aldafermin significantly reduced major hydrophobic bile acids with stronger detergency and cytotoxicity. Our data suggest that bile acids may contribute to maintaining a pro-fibrotic hepatic microenvironment in metabolic and cholestatic liver diseases. In short: Aldafermin is an intestinal hormone analog currently being developed for the treatment of chronic liver disease. This article demonstrates that Aldafermin effectively and stably inhibits toxic hydrophobic bile acids regardless of etiology. Treatment strategies using Aldafermin may be broadly applicable to other chronic gastrointestinal and liver diseases. Clinical trial registration: This study has been registered at Clinicaltrials.gov under registration numbers NCT02443116 and NCT02704364. [4]

C26H43NO4

433.63

433.319

C, 72.02; H, 10.00; N, 3.23; O, 14.76

474-74-8

24404-83-9 (mono-hydrochloride salt)

115245

White to off-white solid powder

1.112g/cm3

619.7ºC at 760mmHg

212-214°C (lit.)

328.6ºC

5.014

3

4

6

31

695

9

C[C@H](CCC(=O)NCC(=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

XBSQTYHEGZTYJE-OETIFKLTSA-N

InChI=1S/C26H43NO4/c1-16(4-9-23(29)27-15-24(30)31)20-7-8-21-19-6-5-17-14-18(28)10-12-25(17,2)22(19)11-13-26(20,21)3/h16-22,28H,4-15H2,1-3H3,(H,27,29)(H,30,31)/t16-,17-,18-,19+,20-,21+,22+,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]acetic acid

Lithocholic acid glycine conjugate; Lithocholylglycine; Glycolithocholic acid; Lithocholylglycine; 474-74-8; Lithocholic acid glycine conjugate; Glycine, N-[(3a,5b)-3-hydroxy-24-oxocholan-24-yl]-; Q53GV75CJG; CHEMBL258818; CHEBI:37998; Glycolithocholic 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)

DMSO : ~50 mg/mL (~115.31 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (2.88 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 12.5 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: ≥ 1.25 mg/mL (2.88 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 12.5 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.)