Lithocholic Acid Concentrations Research Articles

Humid heat environment causes anxiety-like disorder via impairing gut microbiota and bile acid metabolism in mice

Climate and environmental changes threaten human mental health, but the impacts of specific environmental conditions on neuropsychiatric disorders remain largely unclear. Here, we show the impact of a humid heat environment on the brain and the gut microbiota using a conditioned housing male mouse model. We demonstrate that a humid heat environment can cause anxiety-like behaviour in male mice. Microbial 16 S rRNA sequencing analysis reveals that a humid heat environment caused gut microbiota dysbiosis (e.g., decreased abundance of Lactobacillus murinus), and metabolomics reveals an increase in serum levels of secondary bile acids (e.g., lithocholic acid). Moreover, increased neuroinflammation is indicated by the elevated expression of proinflammatory cytokines in the serum and cortex, activated PI3K/AKT/NF-κB signalling and a microglial response in the cortex. Strikingly, transplantation of the microbiota from mice reared in a humid heat environment readily recapitulates these abnormalities in germ-free mice, and these abnormalities are markedly reversed by Lactobacillus murinus administration. Human samples collected during the humid heat season also show a decrease in Lactobacillus murinus abundance and an increase in the serum lithocholic acid concentration. In conclusion, gut microbiota dysbiosis induced by a humid heat environment drives the progression of anxiety disorders by impairing bile acid metabolism and enhancing neuroinflammation, and probiotic administration is a potential therapeutic strategy for these disorders.

A novel near-infrared polymethine dye biosensor for rapid and selective detection of lithocholic acid

Lithocholic acid (LCA), a secondary bile acid, has emerged as a potential early diagnostic biomarker for various liver diseases. In this study, we introduce a novel near-infrared (NIR) polymethine dye-based biosensor, capable of sensitive and selective detection of LCA in phosphate buffer and artificial urine (AU) solutions. The detection mechanism relies on the formation of J-aggregates resulting from the interplay of 3,3-Diethylthiatricarbocyanine iodide (DiSC2(7)) dye molecules and LCA, which induces a distinctive red shift in both absorption and fluorescence spectra. The biosensor demonstrates a detection limit for LCA of 70 μM in PBS solution (pH 7.4), while in AU solution, it responds to an LCA concentration as low as ∼60 μM. Notably, the proposed biosensor exhibits outstanding selectivity for LCA, effectively distinguishing it from common interferents such as uric acid, ascorbic acid, and glucose. This rapid, straightforward, and cost-effective spectrometer-based method underscores its potential for early diagnosis of liver diseases by monitoring LCA concentrations.

Bile acid profiles and mRNA abundance of bile acid-related genes in adipose tissue of dairy cows with high versus normal body condition

Besides their lipid-digestive role, bile acids (BA) influence overall energy homeostasis, such as glucose and lipid metabolism. We hypothesized that BA along with their receptors, regulatory enzymes, and transporters are present in subcutaneous adipose tissue (scAT). In addition, we hypothesized that their mRNA abundance varies with the body condition of dairy cows around calving. Therefore, we analyzed BA in serum and scAT as well as the mRNA abundance of BA -related enzymes, transporters, and receptors in scAT during the transition period in cows with different body conditions around calving. In a previously established animal model, 38 German Holstein cows were divided into either a high (HBCS; n = 19) or normal BCS (NBCS; n = 19) group based on their body condition score (BCS) and back fat thickness (BFT). Cows were fed different diets to achieve the targeted differences in BCS and BFT (NBCS: BCS <3.5, BFT <1.2 cm; HBCS: BCS >3.75, BFT >1.4 cm) until dry-off at 7 wk ante partum. During the dry period and subsequent lactation, both groups were fed the same diets regarding their demands. Using a targeted metabolomics approach via LC-ESI-MS /MS, BA were analyzed in serum and scAT at wk -7, 1, 3, and 12 relative to parturition. In serum, 15 BA (cholic acid (CA), chenodeoxycholic acid (CDCA), glycocholic acid (GCA), taurocholic acid (TCA), glycochenodeoxycholic acid (GCDCA), taurochenodeoxycholic acid (TCDCA), deoxycholic acid (DCA), lithocholic acid (LCA), glycodeoxycholic acid (GDCA), glycolithocholic acid (GLCA), taurodeoxycholic acid (TDCA), taurolithocholic acid (TLCA), β-muricholic acid (MCA(b)), tauromuricholic acid (sum of α and β) (TMCA (a+b)), glycoursodeoxycholic acid (GUDCA)) were observed, whereas in scAT 7 BA (CA, GCA, TCA, GCDCA, TCDCA, GDCA, TDCA) were detected. In serum and scAT samples, the primary BA CA and its conjugate GCA were predominantly detected. Increasing serum concentrations of CA, CDCA, TCA, GCA, GCDCA, DCA, and MCA(b) with the onset of lactation might be related to the increasing DMI after parturition. Furthermore, serum concentrations of CA, CDCA, GCA, DCA, GCDCA, TCA, LCA, and GDCA were lower in HBCS cows compared with NBCS cows, concomitant with increased lipolysis in HBCS cows. The correlation between CA in serum and scAT may point to the transport of CA across cell membranes. Overall, the findings of the present study suggest a potential role of BA in lipid metabolism depending on the body condition of periparturient dairy cows.

Alcohol-induced gut microbiome dysbiosis enhances the colonization of Klebsiella pneumoniae on the mouse intestinal tract.

Chronic alcohol consumption, an important risk factor for diseases and deaths, can cause intestinal microbiota dysbiosis and increase the infection of some opportunistic pathogens. However, the current studies on the effects of alcohol-induced intestinal microbiota dysbiosis on gut colonization of Klebsiella pneumoniae are still scarce. In the present study, we established a binge-on-chronic alcohol model in mice to identify the characteristics of alcohol-induced intestinal microbiome and metabolite dysbiosis using multi-omics and explored the effects and potential mechanisms of these dysbioses on the intestinal colonization of K. pneumoniae. The results show that chronic alcohol consumption alters the diversity and composition of gut microbiota (including bacteria and fungi), decreases the complexity of the interaction between intestinal bacteria and fungi, disturbs the gut metabolites, and promotes the colonization of K. pneumoniae on the gut of mice. The relevance analyses find that alcohol-induced gut microbiome dysbiosis has a strong correlation with the alteration of secondary bile acids. In vitro results suggest that the high concentration of lithocholic acid, a secondary bile acid, could significantly inhibit the proliferation of K. pneumoniae, and the adhesion of K. pneumoniae to Caco-2 cells. Our results indicate that alcohol-induced microbiome dysbiosis contributes to decreased levels of secondary bile acids, which was one of the main reasons affecting the colonization of K. pneumoniae in mice's intestines. Some secondary bile acids (e.g., lithocholic acid) might be a potential drug to prevent the colonization and spread of K. pneumoniae.IMPORTANCEAlcohol is one of the most commonly misused substances in our lives. However, long-term heavy drinking will increase the colonization of some opportunistic pathogens (e.g., Klebsiella pneumoniae) in the body. Here, we revealed that binge-on-chronic alcohol consumption disrupted the balance between gut bacteria and fungi, induced the gut microbiome and metabolites dysbiosis, and promoted the colonization of K. pneumoniae in the intestine of mice. In particular, alcohol-taking disrupted intestinal bile acid metabolism and reduced the lithocholic acid concentration. However, a high concentration of lithocholic acid can protect against intestinal colonization of K. pneumoniae by inhabiting the bacterial growth and adhesion to the host cell. Hence, regulating the balance of gut microbiota and intestinal bile acid metabolism may be a potential strategy for reducing the risk of K. pneumoniae infection and spread.

PSIX-3 A Veterinary Gastrointestinal Low-Fat Diet Affects Fecal Characteristics, Metabolites, Bile Acids, and Microbiota Concentrations of Antibiotic-Treated Dogs

Abstract In a recent dog study, the antibiotic metronidazole proved to be an effective disruptor of the fecal microbiota and resulted in looser stools and altered fecal bile acid (BA) profiles. Recovery took several weeks and was aided by a dietary supplement. The objective of this study was to determine how a veterinary gastrointestinal low-fat diet affected the fecal characteristics and metabolite, BA, and microbiota concentrations of dogs recovering from metronidazole administration. Twenty-four healthy adult dogs were used in an 8-wk completely randomized design study. The study began with a 2-wk baseline phase where all dogs consumed a leading grocery brand dry kibble diet (GBD; wk 0 to wk 2). Over the next 2 wk, dogs continued to consume GBD and received metronidazole (20 mg/kg BW; wk 2 to wk 4). At wk 4, dogs were randomly allotted to one of two treatments [GBD; Blue Buffalo Gastrointestinal Support Low-Fat (BB)] for the remaining 4 wk (wk 5 to wk 8). Fecal scores were recorded daily, and fresh fecal samples were collected at wk 2, 4, 5, 6, 7, and 8 for measurement of pH, dry matter, and metabolite and bile acid concentrations. Fecal microbiota populations were also analyzed by 16S rRNA sequencing and qPCR, which was used to calculate dysbiosis index. All data were analyzed as repeated measures using the Mixed Models procedure of SAS 9.4, with treatment treated as a fixed effect, dog as a random effect, and significance set at P &amp;lt; 0.05. As anticipated, metronidazole administration increased (P &amp;lt; 0.0001) fecal scores (looser stools) and drastically altered (P &amp;lt; 0.05) fecal metabolite concentrations, reducing fecal short-chain fatty acid (SCFA), branched-chain fatty acid (BCFA), phenol, and indole concentrations, increasing primary BA concentrations, and decreasing secondary bile acid concentrations. Metronidazole dramatically impacted fecal microbiota populations by reducing alpha diversity, altering the abundance of multiple bacterial taxa, and increasing dysbiosis index. Fecal outcome variables recovered over the next 4 wk, with some being impacted by diet. The change in fecal SCFA and immunoglobulin A concentrations were greater (P &amp;lt; 0.01), while the change in fecal BCFA, phenol, and indole concentrations were decreased (P &amp;lt; 0.05) in dogs fed BB. Dysbiosis index and alpha diversity measures recovered quickly for all dogs and without dietary effects. Recovery of over 25 bacterial genera were impacted by diet (P &amp;lt; 0.05). While many bacterial taxa returned to baseline levels after 4 wk, others had still not fully recovered. Fecal BA concentrations recovered quickly for all dogs. The change in lithocholic acid concentration was less (P &amp;lt; 0.0001) in dogs fed BB, but other BA concentrations were not affected by diet. In conclusion, metronidazole drastically alters the fecal microbiota and metabolites of dogs. While most variables return to normal over time, a veterinary gastrointestinal diet may aid in the recovery.

Gut microbiota-bile acid-vitamin D axis plays an important role in determining oocyte quality and embryonic development.

To reveal whether gut microbiota and their metabolites are correlated with oocyte quality decline caused by circadian rhythm disruption, and to search possible approaches for improving oocyte quality. A mouse model exposed to continuous light was established. The oocyte quality, embryonic development, microbial metabolites and gut microbiota were analyzed. Intragastric administration of microbial metabolites was conducted to confirm the relationship between gut microbiota and oocyte quality and embryonic development. Firstly, we found that oocyte quality and embryonic development decreased in mice exposed to continuous light. Through metabolomics profiling and 16S rDNA-seq, we found that the intestinal absorption capacity of vitamin D was decreased due to significant decrease of bile acids such as lithocholic acid (LCA), which was significantly associated with increased abundance of Turicibacter. Subsequently, the concentrations of anti-Mullerian hormone (AMH) hormone in blood and melatonin in follicular fluid were reduced, which is the main reason for the decline of oocyte quality and early embryonic development, and this was rescued by injection of vitamin D3 (VD3). Secondly, melatonin rescued oocyte quality and embryonic development by increasing the concentration of lithocholic acid and reducing the concentration of oxidative stress metabolites in the intestine. Thirdly, we found six metabolites that could rescue oocyte quality and early embryonic development, among which LCA of 30mg/kg and NorDCA of 15mg/kg had the best rescue effect. These findings confirm the link between ovarian function and gut microbiota regulation by microbial metabolites and have potential value for improving ovary function.

Diets Enriched in Sugar, Refined, or Whole Grain Differentially Influence Plasma Cholesterol Concentrations and Cholesterol Metabolism Pathways with Concurrent Changes in Bile Acid Profile and Gut Microbiota Composition in ApoE-/- Mice.

This study aimed to compare the effects of diets enriched in sugar, refined grain (RG), or whole grain (WG) on circulating cholesterol concentrations and established and emerging mechanisms regulating cholesterol metabolism. Forty-four male ApoE-/- mice aged 8 weeks were randomly fed an isocaloric sugar-, RG-, or WG-enriched diet for 12 weeks. Compared to WG-enriched diet, fasting plasma LDL-C and HDL-C concentrations were higher and the mRNA expression of intestinal LXR-α was lower in sugar- and RG-enriched diets; plasma TC, non-HDL-C, TG and VLDL-C concentrations, and cecal concentrations of lithocholic acid were higher and the mRNA expression of intestinal ABCG5 was lower in sugar-enriched diet, and the mRNA expression of hepatic IDOL and cecal concentrations of lithocholic and deoxycholic acids was higher in RG-enriched diet. The relative abundance of Akkermansia, Clostridia_UCG-014, Alistipes, and Alloprevotella, which were lower in sugar- and/or RG- than in WG-enriched diet, had inverse correlations with fasting plasma cholesterol concentrations or cecal concentrations of secondary bile acids and positive correlations with gene expressions in intestinal cholesterol efflux. Conversely, the relative abundance of Lactobacillus, Lachnoclostridium, Lachnospiraceae_NK4A136_group, Colidextribacter, and Helicobacter had reverse correlations. Both sugar- and RG-enriched diets had unfavorable effects on cholesterol concentrations; yet, their effects on the gene expressions of cholesterol efflux, uptake, bile acid synthesis, and bile acid concentrations were distinctive and could be partially attributable to the concurrent changes in gut microbiota.

Medium chain fatty acid supplementation improves animal metabolic and immune status during the transition period: A study on dairy cattle.

The transition period is the stage of the high incidence of metabolic and infectious diseases in dairy cows. Improving transition dairy cows' health is crucial for the industry. This study aimed to determine the effects of dietary supplementation medium-chain fatty acids (MCFAs) on immune function, metabolic status, performance of transition dairy cows. Twenty multiparous Holstein cows randomly assigned to two treatments at 35 d before calving. 1) CON (fed the basal 2) MCFA treatment (basal diet was supplemented at an additional 20 g MCFAs mixture every day) until 70 d after calving. The results showed that the serum amyloid A myeloperoxidase concentrations in the blood of cows in MCFA treatment significantly decreased during the early lactation (from 1 d to 28 d after calving) 0.03, 0.04, respectively) compared with the CON, while the tumor necrosis factor concentration was significantly decreased at 56 d after calving (P = 0.02). In addition, the concentration of insulin in the pre-calving (from 21 d before calving to calving) blood of cows in MCFA treatment was significantly decreased (P = 0.04), and concentration of triglyceride also showed a downward trend at 28 d after calving 0.07). Meanwhile, MCFAs supplementation significantly decreased the concentrations of lithocholic acid, hyodeoxycholic acid, and hyocholic acid in the blood at 1 d calving (P = 0.02, < 0.01, < 0.01, respectively), and the level of hyocholic acid taurocholic acid concentrations (P < 0.01, = 0.01, respectively) decreased dramatically at 14 d after calving. However, compared with the CON, the pre-calving dry matter intake and the early lactation milk yield in MCFA treatment were significantly decreased (P = 0.05, 0.02, respectively). In conclusion, MCFAs supplementation transition diet could improve the immune function and metabolic status of dairy cows, and the health of transition cows might be beneficial from the endocrine status.

Vitamin D3 Metabolic Enzymes in Plateau Zokor (Myospalax baileyi) and Plateau Pika (Ochotona curzoniae): Expression and Response to Hypoxia.

Simple SummaryVitamin D3 is mainly synthesized in the skin after exposure to sunlight and plays a fundamental role in the absorption of calcium and phosphate. Lithocholic acid increases intestinal calcium absorption in vitamin D-deficient rats. The plateau zokor, which lives underground throughout its life, and plateau pika are unique animal species on the Qinghai-Tibet Plateau. To investigate how animals that are rarely exposed to sunlight deal with the expected deficit in vitamin D3 levels and the effect of hypoxia on vitamin D3 metabolism, the levels of key biomarkers related to vitamin D3 metabolism, and the mRNA and protein levels of enzymes involved in key pathways were measured. Plateau zokor was deficient in vitamin D3 and has a higher level of lithocholic acid, but its calcium levels were within the normal range. The levels of vitamin D3 and the expression levels of vitamin D3 metabolism-related genes CYP2R1 and CYP27B1 decreased with increasing altitude in plateau pika. Plateau zokor was deficient in vitamin D3, and lithocholic acid appears to substitute it. In addition, hypoxia suppresses vitamin D3 in plateau pika by down-regulating the expression of vitamin D3 metabolism-related genes.Vitamin D3 (D3) is produced endogenously from 7-dehydrocholesterol by irradiation and is an important secosteroid for the absorption of calcium and phosphate. Lithocholic acid (LCA) increases intestinal paracellular calcium absorption in a vitamin D receptor-dependent manner in vitamin D-deficient rats. The plateau zokor (Myospalax baileyi), a strictly subterranean species, and plateau pika are endemic to the Qinghai-Tibet Plateau. To verify whether the zokors were deficient in D3 and reveal the effects of hypoxia on D3 metabolism in the zokors and pikas, we measured the levels of 25(OH)D3, calcium, and LCA, and quantified the expression levels of D3 metabolism-related genes. The results showed an undetectable serum level of 25(OH)D3 and a significantly higher concentration of LCA in the serum of plateau zokor, but its calcium concentration was within the normal range compared with that of plateau pika and Sprague-Dawley rats. With increasing altitude, the serum 25(OH)D3 levels in plateau pika decreased significantly, and the mRNA and protein levels of CYP2R1 (in the liver) and CYP27B1 (in the kidney) in plateau pika decreased significantly. Our results indicate that plateau zokors were deficient in D3 and abundant in LCA, which might be a substitution of D3 in the zokor. Furthermore, hypoxia suppresses the metabolism of D3 by down-regulating the expression of CYP2R1 and CYP27B1 in plateau pika.

Ursodeoxycholic acid production by Gibberella zeae mutants

Ursodeoxycholic acid (UDCA) is a highly demanded pharmaceutical steroid widely used in medicine. An ascomycete Gibberella zeae VKM F-2600 is capable of producing UDCA by 7β-hydroxylation of lithocholic acid (LCA). The present study is aimed at the improvement of the fungus productivity. The original procedures for the protoplast obtaining followed by UV mutagenesis and screening of ketoconazole-resistant mutant clones have been applied. The highest yield of G. zeae protoplasts was obtained when using the mycelium in the active growth phase, ammonium chloride as an osmotic stabilizer and treatment of the fungal cells by the lytic enzymes cocktail from Trichoderma hurzanium. The conditions for effective protoplast regeneration and the UV-mutagenesis were found to provide 6–12% survival rate of the protoplasts with superior number of possible mutations. Three of 27 ketoconazole-resistant mutant clones obtained have been selected due to their increased biocatalytic activity towards LCA. The mutant G. zeae M23 produced 26% more UDCA even at relatively high LCA concentration (4 g/L) as compared with parent fungal strain, and the conversion reached 88% (w/w). The yield of UDCA reached in this study prefers those ever reported. The results contribute to the knowledge on ascomycete mutagenesis, and are of importance for biotechnological production of value added cholic acids.Graphical

Effects of the Probiotic, Lactobacillus delbrueckii subsp. bulgaricus, as a Substitute for Antibiotics on the Gastrointestinal Tract Microbiota and Metabolomics Profile of Female Growing-Finishing Pigs.

Simple SummaryLactobacillus delbrueckii subsp. bulgaricus (LDB) is an important candidate for antibiotic replacement in pig production. In this study, LDB and antibiotic diets were fed to the LDB and antibiotic groups of female growing-finishing pigs, respectively. 16S rRNA sequencing was used to identify different microbiota. Liquid chromatography-mass spectrometry-based non-targeted metabolomics approaches were used to identify different metabolites. The co-occurrence network of the fecal microbiota and metabolite was analyzed. The results contain information on pig growth performance, microbiota data, metabolite data and co-occurrence networks, supporting the possibility of LDB as an antibiotics replacement in pig production.Lactobacillus delbrueckii subsp. bulgaricus (LDB) is an approved feed additive on the Chinese ‘Approved Feed Additives’ list. However, the possibility of LDB as an antibiotic replacement remains unclear. Particularly, the effect of LDB on microbiota and metabolites in the gastrointestinal tract (GIT) requires further explanation. This study aimed to identify the microbiota and metabolites present in fecal samples and investigate the relationship between the microbiota and metabolites to evaluate the potential of LDB as an antibiotic replacement in pig production. A total of 42 female growing-finishing pigs were randomly allocated into the antibiotic group (basal diet + 75 mg/kg aureomycin) and LDB (basal diet + 3.0 × 109 cfu/kg LDB) groups. Fecal samples were collected on days 0 and 30. Growth performance was recorded and assessed. 16S rRNA sequencing and liquid chromatography-mass spectrometry-based non-targeted metabolomics approaches were used to analyze the differences in microbiota and metabolites. Associations between the differences were calculated using Spearman correlations with the Benjamini–Hochberg adjustment. The LDB diet had no adverse effect on feed efficiency but slightly enhanced the average daily weight gain and average daily feed intake (p > 0.05). The diet supplemented with LDB increased Lactobacillus abundance and decreased that of Prevotellaceae_NK3B31_group spp. Dietary-supplemented LDB enhanced the concentrations of pyridoxine, tyramine, D-(+)-pyroglutamic acid, hypoxanthine, putrescine and 5-hydroxyindole-3-acetic acid and decreased the lithocholic acid concentration. The Lactobacillus networks (Lactobacillus, Peptococcus, Ruminococcaceae_UCG-004, Escherichia-Shigella, acetophenone, tyramine, putrescine, N-methylisopelletierine, N1-acetylspermine) and Prevotellaceae_NK3B31_group networks (Prevotellaceae_NK3B31_group, Treponema_2, monolaurin, penciclovir, N-(5-acetamidopentyl)acetamide, glycerol 3-phosphate) were the most important in the LDB effect on pig GIT health in our study. These findings indicate that LDB may regulate GIT function through the Lactobacillus and Prevotellaceae_NK3B31_group networks. However, our results were restrained to fecal samples of female growing-finishing pigs; gender, growth stages, breeds and other factors should be considered to comprehensively assess LDB as an antibiotic replacement in pig production.

Association of Serum and Fecal Bile Acid Patterns With Liver Fibrosis in Biopsy-Proven Nonalcoholic Fatty Liver Disease: An Observational Study.

No reports on both blood and fecal bile acids (BAs) in patients with nonalcoholic fatty liver disease (NAFLD) exist. We simultaneously assessed the serum and fecal BA patterns in healthy participants and those with NAFLD. We collected stool samples from 287 participants from 5 hospitals in Japan (healthy control [HC]: n = 88; mild fibrosis: n = 104; and advanced fibrosis group: n = 95). Blood samples were collected and analyzed for serum BAs and 7α-hydroxy-4-cholesten-3-one (C4)-a surrogate marker for BA synthesis ability-from 141 patients. Concentrations of BAs, including cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid, ursodeoxycholic acid, and lithocholic acid (LCA), were measured using liquid chromatography-mass spectrometry. The total fecal BA concentration was significantly higher in the NAFLD group with worsening of fibrosis than in the HC group. Most of the fecal BAs were secondary and unconjugated. In the fecal BA fraction, CA, DCA, chenodeoxycholic acid, ursodeoxycholic acid, and LCA were significantly higher in the NAFLD than in the HC group. The total serum BA concentration was higher in the NAFLD group with worsening of fibrosis than in the HC group. In the serum BA fraction, CA, LCA, and C4 concentrations were significantly higher in the NAFLD than in the HC group. Fecal and serum BA and C4 concentrations were high in patients with NAFLD with worsening of fibrosis, suggesting involvement of abnormal BA metabolism in NAFLD with fibrosis progression. Abnormalities in BA metabolism may be a therapeutic target in NAFLD with fibrosis.

In vitro protoscolicidal effects of lithocholic acid on protoscoleces of Echinococcus granulosus and its mechanism

Surgery has been found to be the best choice of treatment for hydatidosis. However, leakage of cyst contents during surgery is the foremost reason for recurrence of hydatidosis. In this study, we investigated the in vitro efficacy of lithocholic acid (LCA) against Echinococcus granulosus protoscoleces. The protoscoleces were divided into a control group, an albendazole (ABZ) positive control group and LCA intervention groups at concentrations of 0.5, 1, 2, and 3 mmol/L and stained with 0.1% eosin for observation using an inverted microscope; the protoscolecal ultrastructure was examined with SEM and TEM; the activities of ROS, SOD, and caspase-3 were investigated using an ROS kit, SOD kit, and caspase-3 kit, respectively; the contents of HO-1 and NQO-1 were analyzed by enzyme-linked immunosorbent assay; and the expression level of cytochrome c (Ctyc) was analyzed by western blotting. Results: As the concentration of LCA increased, the survival rate of protoscoleces gradually decreased. The microstructure shows that the external shape and internal structure were gradually deformed and collapse. SOD, GSH, HO-1 and NQO-1 decreased more significantly in the 3 mmol/L LCA group. However, ROS levels gradually increased. LCA treatment for 3 days at all concentrations significantly increased caspase-3 activity and expression in a dose-dependent manner. LCA decreased the level of Ctyc protein in vitro. LCA demonstrated a parasiticidal effect on the protoscoleces of Echinococcus granulosus in vitro. LCA may induce apoptosis of E. granulosus protoscoleces by oxidative stress and mitochondrial pathways.

Taurine Grafted Micro-Implants Improved Functions without Direct Dependency between Interleukin-6 and the Bile Acid Lithocholic Acid in Plasma.

A recent study showed an association between diabetes development and the bile acid lithocholic acid (LCA), while another study demonstrated positive biological effects of the conjugated bile acid, taurocholic acid (TCA), on pancreatic cells. Thus, this study aimed to encapsulate TCA with primary islets (graft) and study the biological effects of the graft, post-transplantation, in diabetic mice, including effects on LCA concentrations. Sixteen mature adult mice were made diabetic and randomly divided into two equal groups, control and test (transplanted encapsulated islets without or with TCA). Graft pharmaceutical features pre-transplantation, and biological effects including on LCA concentrations post-transplantation, were measured. TCA-microcapsules had an oval shape and similar size compared with the control. The treatment group survived longer, showed improved glucose and interleukin-6 concentrations, and lower LCA concentrations in plasma, large intestine, faeces, liver and spleen, compared with control. Results suggest that TCA incorporation with islets encapsulated graft exerted beneficial effects, but there was no direct and significant dependency between concentrations of interleukin-6 and LCA.

1039. Rapid Restoration of Bile Acid Compositions After Treatment with RBX2660 for Recurrent Clostridioides difficile Infection—Results from the PUNCH CD3 Phase 3 Trial

BackgroundMicrobiota-based treatments are increasingly evaluated as a strategy to reduce recurrence of Clostridioides difficile infection (rCDI), and their proposed mechanisms include restoration of the microbiota and microbiota-mediated functions, including bile acid metabolism. RBX2660—a broad-consortium investigational live biotherapeutic—has been evaluated in >600 participants in 6 clinical trials, with consistent reduction of rCDI recurrence. Here we report that fecal bile acid compositions were significantly restored in treatment-responsive participants in PUNCH CD3—a Phase 3 randomized, double-blinded, placebo-controlled trial of RBX2660.MethodsPUNCH CD3 participants received a single dose of RBX2660 or placebo between 24 to 72 hours after completing rCDI antibiotic treatment. Clinical response was the absence of CDI recurrence at eight weeks after treatment. Participants voluntarily submitted stool samples prior to blinded study treatment (baseline), 1, 4 and 8 weeks, 3 and 6 months after receiving study treatment. A liquid chromatography tandem mass spectrometry method was developed to extract and quantify 33 bile acids from all participant fecal samples received up to the 8-week time point. Mean bile acid compositions were fit to a Dirichlet multinomial distribution and compared across time points and between RBX2660- and placebo-treated participants.ResultsClinically, RBX2660 demonstrated superior efficacy versus placebo (70.4% versus 58.1%). RBX2660-treated clinical responders’ bile acid compositions shifted significantly from before to after treatment. Specifically, primary bile acids predominated before treatment, whereas secondary bile acids predominated after treatment (Figure 1A). These changes trended higher among RBX2660 responders compared to placebo responders. Importantly, median levels of lithocholic acid (LCA) and deoxycholic acid (DCA) showed large, significant increases after treatment (Figure 1B). A. Bile acid compositions before (BL) and up to 8 weeks after RBX2660 treatment among treatment responders. Compositions are shown as the fraction of total bile acids classified as primary or secondary conjugated or deconjugated bile acids. B. Concentrations of lithocholic acid (LCA) and deoxycholic acid (DCA) among RBX2660 treatment responders, shown with individual samples and time point group median with interquartile ranges.ConclusionAmong PUNCH CD3 clinical responders, RBX2660 significantly restored bile acids from less to more healthy compositions. These clinically correlated bile acid shifts are highly consistent with results from a prior trial of RBX2660.Disclosures Romeo Papazyan, PhD, Ferring Research Institute (Employee) Bryan Fuchs, PhD, Ferring Pharmaceuticals (Employee) Ken Blount, PhD, Rebiotix Inc., a Ferring Company (Employee)

The inhibition of hepatic Pxr-Oatp2 pathway mediating decreased hepatic uptake of rosuvastatin in rats with high-fat diet-induced obesity

PurposeObesity affecting drug pharmacokinetics results in the risk of the therapeutic failure or toxic side effects of drugs increasing. Unfortunately, the pharmacokinetic data in obese patients still lack for majority of drugs. Therefore, our study principally investigated the effect of obesity induced by high fat-diet (HFD) on the pharmacokinetics of rosuvastatin and explored the underlying mechanism via the hepatic pregnane X receptor (Pxr)- organic anion transporting polypeptide 2 (Oatp2) signaling pathway and multidrug resistance-associated protein 2 (Mrp2) in rats. Main methodsRats with obesity was induced by HFD for 4 weeks, and subsequently, the effect of obesity on the blood concentration, pharmacokinetic parameters and biliary excretion of rosuvastatin administrated intravenously and the hepatic uptake of rosuvastatin in the rat primary hepatocytes were evaluated. Additionally, in order to illuminate the underlying mechanism, the alterations of the mRNA expressions of Oatp2, Mrp2 and Pxr and the concentrations of lithocholic acid (LCA), glycine-LCA (GLCA) and taurine-LCA (TLCA) in liver were determined. Key findingsThe blood concentration of rosuvastatin that has great relationship with the muscle toxicity increased in rats with HFD-induced obesity, which could be principally ascribed to the decreased hepatic uptake of rosuvastatin that was mainly resulted from the inhibition of hepatic Pxr-Oatp2 pathway. SignificanceThe decreased hepatic uptake of rosuvastatin causing the increase of the rosuvastatin concentration in blood under the condition of HFD-induced obesity provides a cue for clinicians to reduce the rosuvastatin dose for obese patients to avoid the occurrence risk of the muscle toxicity of rosuvastatin.

Lithocholic acid inhibits P2X2 and potentiates P2X4 receptor channel gating

The family of ATP-gated purinergic P2X receptors comprises seven bunits (P2X1-7) that are unevenly distributed in the central and peripheral nervous systems as well as other organs. Endogenous modulators of P2X receptors are phospholipids, steroids and neurosteroids. Here, we analyzed whether bile acids, which are natural products derived from cholesterol, affect P2X receptor activity. We examined the effects of primary and secondary bile acids and newly synthesized derivatives of lithocholic acid on agonist-induced responses in HEK293T cells expressing rat P2X2, P2X4 and P2X7 receptors. Electrophysiology revealed that low micromolar concentrations of lithocholic acid and its structural analog 4-dafachronic acid strongly inhibit ATP-stimulated P2X2 but potentiate P2X4 responses, whereas primary bile acids and other secondary bile acids exhibit no or reduced effects only at higher concentrations. Agonist-stimulated P2X7 responses are significantly potentiated by lithocholic acid at moderate concentrations. Structural modifications of lithocholic acid at positions C-3, C-5 or C-17 abolish both inhibitory and potentiation effects to varying degrees, and the 3α-hydroxy group contributes to the ability of the molecule to switch between potentiation and inhibition. Lithocholic acid allosterically modulates P2X2 and P2X4 receptor sensitivity to ATP, reduces the rate of P2X4 receptor desensitization and antagonizes the effect of ivermectin on P2X4 receptor deactivation. Alanine-scanning mutagenesis of the upper halve of P2X4 transmembrane domain-1 revealed that residues Phe48, Val43 and Tyr42 are important for potentiating effect of lithocholic acid, indicating that modulatory sites for lithocholic acid and ivermectin partly overlap. Lithocholic acid also inhibits ATP-evoked currents in pituitary gonadotrophs expressing native P2X2, and potentiates ATP currents in nonidentified pituitary cells expressing P2X4 receptors. These results indicate that lithocholic acid is a bioactive steroid that may help to further unveil the importance of the P2X2, and P2X4 receptors in many physiological processes.

Impact of the gonococcal FC428 penA allele 60.001 on ceftriaxone resistance and biological fitness

ABSTRACT Global dissemination of the Neisseria gonorrhoeae ceftriaxone-resistant FC428 clone jeopardizes the currently recommended ceftriaxone-based first-line therapies. Ceftriaxone resistance in the FC428 clone has been associated with the presence of its mosaic penA allele 60.001. Here we investigated the contribution penA allele 60.001 to ceftriaxone resistance and its impact on biological fitness. Gonococcal isolates expressing penA allele 60.001 and mosaic penA allele 10.001, which is widespread in the Asia-Pacific region and associated with reduced susceptibility to ceftriaxone and cefixime, were genetic engineered to exchange their penA alleles. Subsequent antimicrobial susceptibility analyses showed that mutants containing penA 60.001 displayed 8- to 16-fold higher ceftriaxone and cefixime minimal inhibitory concentrations (MICs) compared with otherwise isogenic mutants containing penA 10.001. Further analysis of biological fitness showed that in vitro liquid growth of single strains and in the competition was identical between the isogenic penA allele exchange mutants. However, in the presence of high concentrations of palmitic acid or lithocholic acid, the penA 60.001-containing mutants grew better than the isogenic penA 10.001-containing mutants when grown as single strains. In contrast, the penA 10.001 mutants outcompeted the penA 60.001 mutants when grown in competition at slightly lower palmitic acid or lithocholic acid concentrations. Finally, the penA 60.001 mutants were outcompeted by their penA 10.001 counterparts for in vivo colonization and survival in a mouse vaginal tract infection model. In conclusion, penA allele 60.001 is essential for ceftriaxone resistance of the FC428 clone, while its impact on biological fitness is dependent on the specific growth conditions.

Comparison of diets enriched in stearic, oleic, and palmitic acids on inflammation, immune response, cardiometabolic risk factors, and fecal bile acid concentrations in mildly hypercholesterolemic postmenopausal women—randomized crossover trial

Direct comparisons between SFAs varying in chain length, specifically palmitic acid (16:0) and stearic acid (18:0), relative to the latter's metabolic product, oleic acid (18:1), on cardiometabolic risk factors are limited. The aim of this study was to determine the relative comparability of diets enriched in palmitic acid, stearic acid, and oleic acid on inflammation and coagulation markers, T lymphocyte proliferation/ex-vivo cytokine secretion, plasma cardiometabolic risk factors, and fecal bile acid concentrations. Hypercholesterolemic postmenopausal women (n=20, mean±SD age 64±7 y, BMI 26.4±3.4 kg/m2, LDL cholesterol≥2.8 mmol/L) were provided with each of 3 diets [55% energy (%E) carbohydrate, 15%E protein, 30%E fat, with ∼50% fat contributed by palmitic acid, stearic acid, or oleic acid in each diet; 5 wk/diet phase] using a randomized crossover design with 2-wk washouts between phases. Outcome measures were assessed at the end of each phase. Fasting LDL-cholesterol and non-HDL-cholesterol concentrations were lower after the stearic acid and oleic acid diets than the palmitic acid diet (all P<0.01). Fasting HDL-cholesterol concentrations were lower after the stearic acid diet than the palmitic acid and oleic acid diets (P<0.01). The stearic acid diet resulted in lower lithocholic acid (P=0.01) and total secondary bile acid (SBA) concentrations (P=0.04) than the oleic acid diet. All other outcome measures were similar between diets. Lithocholic acid concentrations were positively correlated with fasting LDL-cholesterol concentrations (r=0.33; P=0.011). Total SBA, lithocholic acid, and deoxycholic acid concentrations were negatively correlated with fasting HDL cholesterol (r=-0.51 to -0.44; P<0.01) concentrations and positively correlated with LDL cholesterol:HDL cholesterol (r =0.37-0.54; P<0.01) ratios. Dietary stearic acid and oleic acid had similar effects on fasting LDL-cholesterol and non-HDL-cholesterol concentrations and more favorable ones than palmitic acid. Unlike oleic acid, the hypocholesterolemic effect of stearic acid may be mediated by inhibition of intestinal hydrophobic SBA synthesis. These findings add to the data suggesting there should be a reassessment of current SFA dietary guidance and Nutrient Facts panel labeling.This trial was registered at clinicaltrials.gov as NCT02145936.

Potential biomarkers to predict outcome of faecal microbiota transfer for recurrent Clostridioides difficile infection

Background & AimsFaecal microbiota transplantation (FMT) has proven high clinical efficacy in the management of recurrent Clostridioides difficile infection (rCDI) with cure rates of over 80% after a single treatment. Nevertheless, the reasons for failure in the remaining 20% remain elusive. The aim of the present study was to investigate different potential predictors of response to FMT. MethodsFaecal specimens of sixteen patients undergoing FMT for rCDI, as well as samples from the respective donors were collected and analyzed by 16S rRNA gene profiling, bile acid-inducible (baiCD) gene specific qPCR, and liquid chromatography tandem-mass spectrometry (LC–MS/MS) to quantify the concentrations of primary and secondary bile acids. ResultsUsing the faecal concentration of the secondary bile acid lithocholic acid (LCA)within the patient specimens, we were able to predict response to FMT (accuracy 95.2%, sensitivity 100%, specificity 90.9%). By combining the faecal LCA concentration with the urinary pCS concentration, an accuracy of 100% was achieved. ConclusionLCA appears to be a promising marker candidate for prediction of clinical response to FMT. Other makers, such as urinary concentration of pCS, but not 3-IS, might be used to improve accuracy of prediction. Further studies are warranted to validate these candidate markers.