Bile Acid Research Articles

8236 Hepatocyte Vitamin D Receptor (VDR) Regulates Liver Regeneration

Abstract Disclosure: J.E. Gunton: None. Harendran Elangovan [1], Rebecca A. Stokes [1], Jeremy Keane [1], Sarinder Chahal [1], Caroline Samer [1], Miguel Agoncillo [1], Josephine Yu [1], Jennifer Chen [1], Michael Downes 2, Ronald M. Evans 2, Christopher Liddle [1],2, Jenny E. Gunton [1] [1]Centre for Diabetes, Obesity and Endocrinology, The Westmead Institute for Medical Research, University of Sydney, Westmead, NSW, Australia 2 Salk Institute for Biological Studies, La Jolla, CA. USA Background: Vitamin D signals through the vitamin D receptor (VDR) to induce its end-organ effects. Hepatic stellate cells control development of liver fibrosis in response to stressors and vitamin D signaling decreases fibrogenesis. VDR expression in hepatocytes, however, is low in healthy liver, and the role of VDR in hepatocyte proliferation is unclear. Methods: In these studies, hepatocyte-VDR null mice (hVDR) were used to assess the role of VDR and vitamin D signaling in hepatic regeneration. Mice underwent 2/3 partial hepatectomy and liver regeneration and function were studied. Results: hVDR mice had impaired liver regeneration with impaired hepatocyte proliferation. This was associated with significant differential changes in bile salts. Notably, mice lacking hepatocyte VDR had significant increases in expression of conjugated bile acids after partial hepatectomy, consistent with failure to normalize hepatic function by the 14-day time point tested. CDCA (chenodeoxycholate) is an FXR ligand, which was increased after surgery in controls but not in hVDR mice. FXR signaling is important for hepatocyte proliferation. Real-time PCR of hVDR and control livers showed significant changes in expression of cell cycle genes including cyclins D1 and E1 and cyclin-dependent kinase 2. Gene expression profiling of hepatocytes treated with vitamin D or control showed regulation of groups of genes involved in liver proliferation, hepatitis, liver hyperplasia / hyperproliferation and liver necrosis / cell death. Conclusions: Together these studies demonstrate an important functional role for VDR in hepatocytes during liver regeneration. Combined with the known profibrotic effects of impaired VDR signaling in stellate cells, these studies provide a mechanism whereby vitamin D deficiency would both reduce hepatocyte proliferation and permit fibrosis, leading to significant liver compromise. Presentation: 6/2/2024

A multi-omics approach reveals the changes in the gut microbiome and metabolism of large yellow croaker (Larimichthys crocea) by dietary supplementation with Bacillus

The intestine serves as the primary site for nutrient absorption in animals. Research in gut biology is increasingly focused on understanding its correlation with the microbiome, specifically by employing probiotics in diets to enhance gut health and stimulate animal growth. In this study, we isolated two Bacillus species (Bacillus halophilus and Lysinibacillus sphaericus) from the intestinal tract of a large yellow croaker and investigated their roles as dietary supplements, assessing their impact on intestinal microbial composition and their efficacy in large yellow croaker growth and immunity. These functions are intrinsically linked to the growth and immune response of large yellow croaker. Our investigation included a comprehensive analysis of various omics results derived from intestinal content samples, encompassing 16S rRNA sequencing and non-targeted metabolomics. These analyses were combined with assessments of differences in immune functionalities observed in the liver and head kidney, aiming to understand the interaction between the two Bacillus species, intestinal microorganisms, and host metabolism. Our findings demonstrated several significant outcomes: (1) Supplementation with two Bacillus species induced considerable alterations in the intestinal microbiome, particularly in the suppression of Vibrionaceae among juvenile large yellow croaker. (2) The influence of pathogenic bacteria such as Vibrio on intestinal health is not solely associated with their characteristics but also closely linked to their relative abundance in the intestine. (3) Distinct dietary groups further influenced changes in the metabolome profile by impacting the intestinal microbiota structure in juvenile large yellow croaker, thereby influencing various metabolites such as amino acids, terpenes, bile acids, and steroids. (4) Differences in the composition of intestinal microbiota among different feed types significantly affected individual antioxidant functionalities, impacting the morphology and immune capabilities of immune organs like the liver. Exploring intricate host-microbiome interactions using multi-omics approaches can provide a better assessment of probiotics' functional potential. Our results revealed that Lysinibacillus sphaericus enhanced intestinal development, increased antioxidant enzyme activity, and facilitated the growth of large yellow croaker.

Antibiotic Resistance Pattern of Lactobacillus reuteri, Lactobacillus salivarius, and Enterococcus hirae Isolated from Gastrointestinal and Respiratory Tract in Commercial Broiler Chickens

Ever since antibiotics were discovered for the treatment of bacterial diseases, an increase in antibiotic-resistant bacteria has been noticed as an economic and public health concern with a high rate of morbidity and mortality. Antibiotic resistance in broiler chickens can be a great threat to public health. This research aimed to screen commercial poultry’s gastrointestinal and respiratory tract bacteria to observe the antibiotic resistance pattern. In this experiment, gastrointestinal tract (GIT) and respiratory tract (RT) bacteria were identified using 16s rRNA gene sequencing from broiler chickens in anaerobic conditions following a bile salt tolerance assay and an antibiotic susceptibility test. Lactobacillus reuteri, Lactobacillus salivarius, and Enterococcus hirae were identified as the isolates. The bacteria found in the gut had a modest degree of bile salt tolerance. The isolates were sensitive to amoxicillin, gentamicin, and streptomycin but resistant to tetracycline, levofloxacin, metronidazole, azithromycin, and erythromycin. This study ensures that the GIT and RT of broiler chicken is a hidden source of the isolated bacteria. A large part of this bacterial population is antibiotic-resistant and reported as a public health concern.

6826 Mevalonate Treatment Worsened Hypocholesterolemia During Prolonged Sepsis Without Affecting Adrenal And Muscle Function

Abstract Disclosure: L. De Bruyn: None. S. Derde: None. F. Van Beek: None. S. Vander Perre: None. I. Derese: None. L. Pauwels: None. G. Van den Berghe: None. L. Langouche: None. Introduction: Sepsis is hallmarked by an immediate and sustained reduction in total-, HDL- and LDL-cholesterol (-C), associated with illness severity and a potential contributor to the development of adrenal failure and muscle weakness. The underlying mechanisms that might be involved in sepsis-induced hypocholesterolemia are multifactorial and partly understood. We hypothesized that treatment with the cholesterol precursor mevalonate might improve cholesterol availability, thereby improving adrenal gland and skeletal muscle function. Our hypothesis was tested in a validated and clinically relevant mouse model of prolonged sepsis. Methods: In a catheterized mouse model of cecal ligation and puncture-induced, fluid resuscitated and antibiotics treated polymicrobial abdominal sepsis (5 d), septic mice received continuous infusion with either mevalonate (78 mg/d) or placebo solution, whereas healthy mice served as controls (N=50). Plasma total-C (HDL- + LDL-C), CORT and total bile acids were measured, in addition to ex vivo muscle force and adrenocortical cholesterol ester content. Gene expression markers of inflammation (Tnf-α, Il-6) were measured in the adrenal gland, as well as hepatic mRNA expression of cholesterol synthesis enzymes (Hmgcs1, Hmgcr, Fdft1). Metabolites of the mevalonate pathway (mevalonic acid, squalene, lanosterol, desmosterol and (7-dehydro) cholesterol) were quantified in the liver with LC-MS. Results: The sepsis-induced reduction in plasma total-C was further decreased in mevalonate-treated septic mice (26±3 mg/dL) as compared with placebo (53±3 mg/dL) and healthy controls (108±3 mg/dL) (p<0.0001). Metabolites of the mevalonate pathway were all increased in the liver after mevalonate treatment (p<0.01), whereas hepatic cholesterol content was decreased as compared with placebo (p<0.05). Also, gene expression markers of cholesterol synthesis enzymes were further decreased in the liver of mevalonate-treated septic mice as compared with placebo (p<0.05). In the context of the unexpected further reduction in plasma total-C, no additional effect on plasma CORT, total bile acids, and sepsis-induced loss of muscle force and depletion in adrenocortical cholesterol ester content was observed. In addition, the sepsis-induced rise in markers of adrenal inflammation (Tnf-α, Il-6) was not further affected by mevalonate treatment. Conclusion: Mevalonate treatment further reduced the sepsis-induced hypocholesterolemia in prolonged septic mice, potentially due to increased feedback inhibition on hepatic cholesterol biosynthesis, but without further affecting adrenal and muscle function. Other therapies such as substitution with bovine serum HDL-C are currently investigated to improve altered cholesterol availability during sepsis. Presentation: 6/2/2024

Association of Serum Bile Acid Profile with Diet and Physical Activity Habits in Japanese Middle-Aged Men.

Circulating bile acid (BA) profiles change with lifestyle and are closely related to intestinal BA metabolisms such as deconjugation and conversion to secondary BAs. The composition of BA in the blood is involved in systemic nutrient metabolism and intestinal health. Herein, we explored the associations of lifestyle and physical fitness with the circulating BA profile of middle-aged men. Data of 147 male participants (aged 50-64 years; BMI < 26 kg/m2; no medication for diabetes or dyslipidemia) from the Japan Multi-Institutional Collaborative Cohort study were analyzed. Serum concentrations of 15 types of BAs were examined for associations with variables on dietary habits, physical-activity habits, and physical fitness. Green tea intake was positively associated with the deconjugation ratio of total BAs (p = 0.028) and negatively associated with secondary BA levels (free deoxycholic acid [DCA] (p = 0.078), glyco-DCA (p = 0.048), and tauro-DCA (p = 0.037)). In contrast, physical activity was negatively associated with the deconjugation ratio (p = 0.029) and secondary BA levels (free DCA (p = 0.098), and free lithocholic acid (p = 0.009)). Grip strength was also negatively associated with secondary BA levels (tauro-DCA (p = 0.041)) but was not associated with the deconjugation ratio. Energy and fat intake and skeletal muscle mass were not associated with the deconjugation ratio or secondary BA levels. The study findings suggest that lifestyle-associated changes in serum deconjugated and secondary BAs indicate improvements in nutrient metabolism and the intestinal environment.

Hepatic FXR-FGF4 is required for bile acid homeostasis via an FGFR4-LRH-1 signal node under cholestatic stress.

Bile acid (BA) homeostasis is vital for various physiological processes, whereas its disruption underlies cholestasis. The farnesoid X receptor (FXR) is a master regulator of BA homeostasis via the ileal fibroblast growth factor (FGF)15/19 endocrine pathway, responding to postprandial or abnormal transintestinal BA flux. However, the de novo paracrine signal mediator of hepatic FXR, which governs the extent of BA synthesis within the liver in non-postprandial or intrahepatic cholestatic conditions, remains unknown. We identified hepatic Fgf4 as a direct FXR target that paracrinally signals to downregulate Cyp7a1 and Cyp8b1. The effect of FXR-FGF4 is mediated by an uncharted intracellular FGF receptor 4 (FGFR4)-LRH-1 signaling node. This liver-centric pathway acts as a first-line checkpoint for intrahepatic and transhepatic BA flux upstream of the peripheral FXR-FGF15/19 pathway, which together constitutes an integral hepatoenteric control mechanism that fine-tunes BA homeostasis, counteracting cholestasis and hepatobiliary damage. Our findings shed light on potential therapeutic strategies for cholestatic diseases.

Bile Acids-Based Therapies for Primary Sclerosing Cholangitis: Current Landscape and Future Developments.

Primary sclerosing cholangitis (PSC) is a rare, chronic liver disease with no approved therapies. The ursodeoxycholic acid (UDCA) has been widely used, although there is no evidence that the use of UDCA delays the time to liver transplant or increases survival. Several candidate drugs are currently being developed. The largest group of these new agents is represented by FXR agonists, including obeticholic acid, cilofexor, and tropifexor. Other agents that target bile acid metabolism are ASTB/IBAP inhibitors and fibroblasts growth factor (FGF)19 analogues. Cholangiocytes, the epithelial bile duct cells, play a role in PSC development. Recent studies have revealed that these cells undergo a downregulation of GPBAR1 (TGR5), a bile acid receptor involved in bicarbonate secretion and immune regulation. Additional agents under evaluation are PPARs (elafibranor and seladelpar), anti-itching agents such as MAS-related G-protein-coupled receptors antagonists, and anti-fibrotic and immunosuppressive agents. Drugs targeting gut bacteria and bile acid pathways are also under investigation, given the strong link between PSC and gut microbiota.

Novel sodium tauroursodeoxycholate-based multifunctional liposomal delivery system for encapsulation of oleanolic acid and combination therapy of type 2 diabetes mellitus

Liposomes have demonstrated great potential for drug delivery and diabetes treatment. However, hydrolysis by enzymes and emulsification by endogenous bile salts make liposomes unstable in the gastrointestinal tract. In this study, sodium tauroursodeoxycholate (TUDCNa)-based multifunctional bilosomes were designed to address the deficiencies of conventional liposomes. In the designed bilosomes, cholesterol was replaced by TUDCNa, which served as both a membrane stabilizer and an antidiabetic drug. Oleanolic acid (OA) was encapsulated in both conventional liposomes (OA-Ch-Lip) and bilosomes (OA-Tu-Bil) to compare their properties. Firstly, OA-Tu-Bil exhibited similar encapsulation efficiency and drug loading compared to OA-Ch-Lip, but with a smaller particle size. Secondly, OA-Tu-Bil showed better stability than OA-Ch-Lip. Thirdly, bilosomes exhibited prolonged intestinal retention time and improved permeability and oral bioavailability. Fourthly, in type 2 diabetes mellitus (T2DM) mice model, TUDCNa synergized with OA to exhibit the strongest therapeutic effect. In conclusion, TUDCNa have demonstrated the ability to substitute cholesterol in conventional liposomes, it provided a new approach for oral delivery of hypoglycemic drugs, and offered an innovative strategy for combination therapy.

Effects of complex probiotics on intestinal function and its regulatory mechanism in patients with constipation.

Chronic constipation is a multi-symptomatic, multifactorial, and heterogeneous gastrointestinal disorder. Current pharmacological treatments for chronic constipation are limited and might negatively impact the patients' quality of life. Although probiotics have been shown to improve constipation symptoms, their specific regulatory mechanisms remain unclear. This study sought to explore how probiotic complexes may affect chronic constipation by improving patients' defecation habits. Furthermore, microbial profiles and non-targeted metabolites were assessed to explore the metabolic pathways involved in the improvement of constipation by probiotics. Patients with chronic constipation were treated using a single-blind, randomised, placebo-controlled trial design. The experimental group was administered Lactobacillus powder prepared from 15 probiotic products, and maltodextrin was used as a placebo. Samples were collected twice daily for 4 weeks, and faecal samples were analysed using 16S rRNA sequencing and untargeted metabolic histology. Probiotic treatment changed the makeup of the gut microbiota, enhanced the quantity of Bifidobacterium and Lactobacillus, and markedly reduced clinical symptoms. The 16S rRNA analysis revealed that the abundance of Bifidobacterium and Prevotella increased while that of Thickettsia declined. Moreover, there was a decrease in the abundance of Faecalibacterium and Roseburia. Non-targeted metabolomics analysis identified several differential metabolites, including succinic acid, fumaric acid, cholesterol, xanthurenic acid, 3-alpha,7-alpha-trihydroxy-5beta-cholestan-26-oic, and N-methyltryptamine. KEGG analysis showed that these metabolites were mainly associated with metabolic pathways such as primary bile acid biosynthesis, tryptophan metabolism, alanine, aspartate and glutamate metabolism, phenylalanine metabolism, cholesterol metabolism, and propanoate metabolism. In this study, gut microbiome and non-targeted metabolome analyses were performed on collected faecal samples to compare characteristic microorganisms and differential metabolites to provide new insights and references for probiotic intervention in constipation. Trial registered at chictr.org.cn under number: ChiCTR2200056274.

Oral exposure to benzalkonium chlorides in male and female mice reveals alteration of the gut microbiome and bile acid profile.

Benzalkonium chlorides (BACs) are commonly used disinfectants in a variety of consumer and food-processing settings, and the COVID-19 pandemic has led to increased usage of BACs. The prevalence of BACs raises the concern that BAC exposure could disrupt the gastrointestinal microbiota, thus interfering with the beneficial functions of the microbes. We hypothesize that BAC exposure can alter the gut microbiome diversity and composition, which will disrupt bile acid homeostasis along the gut-liver axis. In this study, male and female mice were exposed orally to d7-C12- and d7-C16-BACs at 120 µg/g/day for one week. UPLC-MS/MS analysis of liver, blood, and fecal samples of BAC-treated mice demonstrated the absorption and metabolism of BACs. Both parent BACs and their metabolites were detected in all exposed samples. Additionally, 16S rRNA sequencing was carried out on the bacterial DNA isolated from the cecum intestinal content. For female mice, and to a lesser extent in males, we found that treatment with either d7-C12- or d7-C16-BAC led to decreased alpha diversity and differential composition of gut bacteria with notably decreased actinobacteria phylum. Lastly, through a targeted bile acid quantitation analysis, we observed decreases in secondary bile acids in BAC-treated mice, which was more pronounced in the female mice. This finding is supported by decreases in bacteria known to metabolize primary bile acids into secondary bile acids, such as the families of Ruminococcaceae and Lachnospiraceae. Together, these data signify the potential impact of BACs on human health through disturbance of the gut microbiome and gut-liver interactions.

The impact of host genetics on microbe assemblages in three types of mrigal carp (Cirrhinus mrigala)

Host–microbiota interactions are molecular and physical interactions between the microbiota and the host that play significant roles in the lifespan of animals. In the present study, two types of gynogenetic mrigal carp (GMCC and GMCW) and the original maternal mrigal carp (MC) were used as a models to investigate host–microbiota interactions. The two types of gynogenetic mrigal carps showed faster growth and better cold tolerance than the MC fish. Compared to those in MC, a large number of goblet cells and higher activity of cellulase and pepsin were detected in the intestine of gynogenetic fish. The composition and abundance of the microbial communities significantly differed among the three types of mrigal carp and water, with Proteobacteria, Firmicutes and Chloroflexi being the most dominant microbes in the GMCC, GMCW and MC fish, respectively. Pseudomonas, Lactococcus and Defluviicoccus were the biomarker genera in the GMCC, GMCW and MC fish. Network analysis revealed no relationships between fish and water, but the dominant genera was positively correlated with the abundance of certain enzymes. Functional analysis revealed the dominant genera associated with amino acid metabolism, fatty acid metabolism and bile acid biosynthesis. Our results suggested that host genetics may affected gut microbe assembly and that the specific metabolic functions of gut microbes may contribute to growth performance through the microbe-gut-liver axis.

Sensitivity of dairy calf Salmonella enterica serotype Cerro isolates to infection-relevant stressors.

Salmonella enterica serotype Cerro (S. Cerro) is an emerging Salmonella serotype isolated from cattle, but the association of S. Cerro with disease is not well understood. While comparative genomic analyses of bovine S. Cerro isolates have indicated mutations in elements associated with virulence, the correlation of S. Cerro fecal shedding with clinical disease in cattle varies between epidemiologic studies. The primary objective of this study was to characterize the infection-relevant phenotypes of S. Cerro fecal isolates obtained from neonatal calves born on a dairy farm in Wisconsin, USA. The S. Cerro isolates varied in biofilm production and sensitivity to the bile salt deoxycholate. All S. Cerro isolates were sensitive to sodium hypochlorite, hydrogen peroxide, and acidic shock. However, S. Cerro isolates were resistant to nitric oxide stress. Two S. Cerro isolates were unable to compete with S. Typhimurium during infection of calf ligated intestinal loops, indicating decreased fitness in vivo. Together, our data suggest that S. Cerro is sensitive to some innate antimicrobial defenses present in the gut, many of which are also used to control Salmonella in the environment. The observed phenotypic variation in S. Cerro isolates from a single farm suggest phenotypic plasticity that could impact infectious potential, transmission, and persistence on a farm.IMPORTANCESalmonella enterica is a zoonotic pathogen that threatens both human and animal health. Salmonella enterica serotype Cerro is being isolated from cattle at increasing frequency over the past two decades; however, its association with clinical disease is unclear. The goal of this study was to characterize infection-relevant phenotypes of S. Cerro isolates obtained from dairy calves from a single farm. Our work shows that there can be variation among temporally related S. Cerro isolates and that these isolates are sensitive to killing by toxic compounds of the innate immune system and those used for environmental control of Salmonella. This work contributes to our understanding of the pathogenic potential of the emerging pathogen S. Cerro.

Sulforaphane Improves Liver Metabolism and Gut Microbiota in Circadian Rhythm Disorder Mice Models Fed With High-Fat Diets.

This study aims to investigate the effect of sulforaphane (SFN) on hepatic metabolism and gut microbiota in a shifted circadian rhythm (CR) mouse model fed with a high-fat diet (HFD). A shifted CR mouse model with HFD is constructed. Biochemical analyses are used to evaluate the effects of SFN on lipid accumulation and liver function. Targeted metabolomics is used for liver metabolites. Results from hematoxylin and eosin staining and Oil Red O staining show that SFN improves liver lipid accumulation and intestinal inflammatory damage in shifted CR treatment with HFD. The concentrations of amino acid metabolites are increased, and the levels of bile acid metabolites are significantly decreased by SFN treatment. Results from 16S rRNA gene sequencing indicate that SFN modulates gut microbiota, particularly by enhancing beneficial bacteria such as Lachnospiraceae, Lactobacillus, Alistipes, Akkermansia, and Eubacteriaum coprostanoligenes. Correlation analysis confirms a close relationship between intestinal microbiota and hepatic metabolites. SFN significantly regulates CR protein expression in the hypothalamus and liver tissues. SFN alleviates hepatic metabolic disorder and gut microbiota dysbiosis induced by CR disruption under a high-fat diet in a mouse model, indicating the potential of SFN in regulating CR disruption.

Influence of bile salts on the gastrointestinal digestion of agar-casein hybrid systems and the nanoassembly of their digestion products

This manuscript reports on the effect of different bile salts concentration on the gastrointestinal digestion of casein and casein-agar hybrid systems and evaluates the effect on the nanostructural assembly of the digestion products through the application of advanced small angle X-ray scattering analysis (SAXS).The results showed that bile salts promote the hydrolysis of micellar casein upon in vitro gastrointestinal digestions. It is noteworthy that in the presence of agar, bile salts interact with the polysaccharide, leading to a reduction in their proteolytic activity. While structured agar-casein hydrogels were shown to be able to limit the proteolysis during the gastric phase, increasing concentrations of bile salts promoted the diffusion of casein from the gel network during the intestinal phase, hence leading to higher degree of hydrolysis. The released digestion products were seen to interact with bile salts, forming ordered lamellar/micellar nanostructures. In particular, the presence of solubilized agar in the digesta was seen to promote the formation of these nanostructures. These nanostructures were formed at 10 mM bile salts for the hydrogels, and across all concentrations for agar-casein blends. Thus, this work demonstrates the great relevance of bile salts in the nanostructural assembly of protein digestion products, which is expected to have a great relevance in bioavailability and metabolic responses induced by protein-rich foods.

Elevated plasma bile acids coincide with cardiac stress and inflammation in young Cyp2c70-/- mice.

High plasma bile acids (BAs), for instance due to intrahepatic cholestasis of pregnancy or neonatal cholestasis, are associated with cardiac abnormalities. Here, we exploited the variability in plasma BA levels in Cyp2c70-/- mice with a human-like BA composition to investigate the acute effects of elevated circulating BAs on the heart. RNA sequencing was performed on hearts of 3-week-old Cyp2c70-/- mice lacking mouse-specific BA species that show features of neonatal cholestasis. Cardiac transcriptomes were compared between wild-type pups, Cyp2c70-/- pups with low or high plasma BAs, and Cyp2c70-/- pups from dams that were perinatally treated with ursodeoxycholic acid (UDCA). We identified 1355 genes that were differentially expressed in hearts of Cyp2c70-/- mice with high versus low plasma BAs with enrichment of inflammatory processes. Strikingly, expression of 1053 (78%) of those genes was normalized in hearts of pups of UDCA-treated dams. Moreover, 645 cardiac genes strongly correlated to plasma BAs, of which 172 genes were associated with cardiovascular disease. Elevated plasma BAs alter gene expression profiles of hearts of mice with a human-like BA profile, revealing cardiac stress and inflammation. Our findings support the notion that high plasma BAs induce cardiac complications in early life. Cyp2c70-/- mice with a human-like bile acid composition show features of neonatal cholestasis but the extrahepatic consequences hereof have so far hardly been addressed Elevated plasma bile acids in Cyp2c70-/- pups coincide with cardiac stress and inflammation Perinatal treatment with UDCA prevents dysregulated cardiac gene expression patterns in Cyp2c70-/- pups.

Total Iridoid Glycosides from Swertia mussotii Franch. Alleviate Cholestasis Induced by α-Naphthyl Isothiocyanate through Activating the Farnesoid X Receptor and Inhibiting Oxidative Stress

Cholestasis refers to a physiological and pathological process caused by bile acid (BA) overaccumulation inside the circulatory system and liver, leading to systemic and hepatocellular damage. Activating the farnesol X receptor (FXR) to restore BA homeostasis is a promising strategy for treating cholestasis. The objective of this research is to reveal solid evidence for the fact that the total iridoid glycosides from Swertia mussotii Franch. (IGSM) alleviate cholestasis. In this research, the whole plant of S. mussotii was extracted with 70% ethanol and separated by macroporous adsorption resin. A rat cholestasis model was established by the injection of α-naphthyl isothiocyanate (ANIT) at a dose of 75 mg/kg. Biochemical and oxidative stress indicators were determined using commercial assay kits. The mRNA abundance of FXR and target proteins was assessed using RT-qPCR. In addition, the effects of main compounds with FXR were evaluated by molecular docking after IGSM analysis using UPLC. The results indicated that IGSM alleviated ANIT-induced cholestasis through reducing serum ALT, AST, AKP, and TBA levels; increasing the mRNA levels of Fxr, Besp, Ntcp, and Mep2; and reducing oxidative stress. The proportion of iridoid compounds in IGSM exceeded 50%, which may be the active substance basis of IGSM. This study provides a theoretical reference for IGSM in the treatment of cholestasis, and future studies may delve more deeply into the FXR regulatory pathway.

A-340 Analysis of OrganOx Liver Perfusate at the Point-of-Care on the Abbott iSTAT and Nova Glucometer to Support Clinical Evaluations of Donor Livers Prior to Transplantation

Abstract Background The recently FDA-approved OrganOx Metra normothermic perfusion system for maintaining donor livers under physiologic conditions prior to transplantation comes equipped with a limited onboard blood gas analyzer for monitoring pO2, pCO2 and temperature. In clinical practice, however, additional biomarkers are used to assess the functional performance of the donor liver, including perfusate lactate clearance and glucose utilization. Liver perfusate contains a mixture of packed red blood cells, albumin, heparin, antibiotic, and solutions of amino acids and electrolytes and bile salts to support metabolic function, and is a specimen type that is not approved by the FDA. Implementation of the OrganOx Metra at Loyola University Medical Center (LUMC), a quaternary care transplant center, led to the validation of liver perfusate for the evaluation of glucose and lactate at the point-of-care (POC) using the Nova STATStrip Glucometer and Abbott iSTAT CG4 cartridge, respectively. Methods Lactate was measured on the Abbott iStat CG4 cartridge, and glucose which was measured using the Nova STATStrip Glucometer. Recovery studies were performed by mixing liver perfusate (non-centrifuged, n=16 independent specimen) with heparinized whole blood. Results The average recovery for the evaluation of lactate was 100% (range = 78 to 122%), and the average recovery for glucose was 109% (range = 93 to 112%). Conclusions Recovery studies evaluating matrix effect for the analysis of lactate (Abbott iSTAT, CG4) and glucose (Nova STATStrip) in liver perfusate indicate that the matrix of the perfusate does not affect the analytical accuracy, as compared to heparinized whole blood, the FDA-approved specimen type. Measurement of the OrganOx liver perfusate for lactate and glucose on these POC devices demonstrates reliable assessments to guide transplant teams in evaluating the viability of donor livers.

B-034 Performance Evaluation of the Total Bile Acids (TBA) Assay on Mindray BS-2800M Analyzer

Abstract Background Bile acids (BA) are synthesized in the liver and secreted into bile. The function of BA is to promote absorption of lipids including fat-soluble vitamins. During liver diseases such as viral hepatitis and liver injury by hepatotoxic drugs, and cholestasis condition, BA secretion to bile is impaired, causing serum BA level to rise. Therefore, the determination of serum BA can be used as a sensitive indicator of liver function. TBA reagent contains NADH that requires pH ≥ 9 to be stable that is usually difficult to be maintained during reagent opened and stored on the analyzer. Meanwhile, 3α-Hydroxysteroid Dehydrogenase (3α-HSD) is required for the reaction. 3α-HSD is sensitive to pH condition. Minor changes in pH results in significant changes of enzyme activity. We formulated our TBA reagent with prolonged stability when reagent is onboard on the analyzer. This study evaluated the performance of the new Mindray TBA assay on Mindray BS-2800M. Methods TBA assay was based on the method with coupled Thio-NAD and NADH reaction for BA. The R1 contains Thio-NAD. The R2 contains NADH and 3α-HSD. R1 is incubated with serum sample containing BA for 5 min at 37°C and R2 is then added. During further incubation in the presence of Thio-NAD, 3α-HSD converts BA to 3-ketosteroids and Thio-NADH. The reaction is reversible, and 3α-HSD can convert 3-keto steroids and Thio-NADH to BA and Thio-NAD. In the presence of excess NADH, enzyme cycling occurs efficiently and the rate of formation of Thio-NADH is determined by measuring specific change of absorbance at 412 nm. Two-point-line calibration is used by this assay. The TBA concentrations for the test samples are obtained by the absorbances of test samples from the calibration line. The performances of precision, linearity, onboard stability, interference (hemolysis, lipid and bilirubin), correlation are evaluated following CLSI protocols. Results The precision study was run according to EP05A3 with two commercial controls (about 21 and 36μmol/L) and two serum sample pools(about 9.9 and 24.2 μmol/L)on the Mindray BS-2800M system. The repeatability and within-lab CVs ranged from 0.74% to 1.97%, respectively. The linearity of the assay was determined as from 2.0 μmol/L to 180 μmol/L. The reagent onboard stability is for 28 days without recalibration that is superior to TBA reagents from other manufacturers. The interference of hemolysis, lipid and bilirubin are with less than ±10% impact with hemolysate Hemoglobin up to 500 mg/dL, Intralipids up to 500 mg/dL, and Conjugated bilirubin up to 30 mg/dL, Unconjugated bilirubin up to 30mg/dL. This assay (Y) also correlated well with SEKISUI TBA assay (x), (Y = 0.9965X + 0.2694, r= 0.9993). Conclusions From this evaluation, we concluded that Mindray TBA assay can measure serum TBA precisely and accurately with good performances, especially for onboard stability. It is suitable for use in routine clinical laboratories.

Explore the mechanism of yishenjiangya formula in the treatment of senile hypertension based on multi-omics technology

Ethnopharmacological relevanceThe Yishenjiangya formula (YSJ) is a traditional Chinese medicine (TCM) primarily composed of qi-tonifying components. This classic formula is commonly utilized to treat kidney qi deficiency in elderly patients with hypertension. According to TCM, maintaining a balance between qi and blood is crucial for stable blood pressure. Kidney qi deficiency can disrupt this balance, altering fluid shear force and, ultimately, leading to hypertension, particularly in elderly populations. Despite YSJ's efficacy in treating hypertension, its specific anti-hypertensive mechanisms remain unclear. Aim of the studyYSJ is commonly prescribed for elderly patients with hypertension. Earlier metabolomics studies demonstrated that YSJ exerts antihypertensive effects by influencing four key pathways: linoleic acid metabolism, glycerol phospholipid metabolism, arginine and proline metabolism, and steroid hormone biosynthesis. This study aims to combine metabolomic and proteomic analyses to thoroughly understand the molecular biological mechanisms responsible for YSJ's anti-hypertensive properties. MethodsUltra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) metabolomics, combined with Label-Free Quantitation (LFQ) proteomics, was employed to analyze serum samples from elderly individuals with and without hypertension pre- and post-YSJ intervention. Serum levels of candidate proteins were assessed using enzyme-linked immunosorbent assay, and receiver operating characteristic curves were used to evaluate the diagnostic performance of the target proteins. ResultsEight differentially expressed metabolites and three differentially expressed proteins were identified as potential therapeutic targets of YSJ. These substances are primarily involved in unsaturated fatty acid metabolism, fluid shear stress and atherosclerosis pathway, primary bile acid biosynthesis, proline metabolism, apoptosis, and endoplasmic reticulum stress. YSJ exerts its therapeutic effects on hypertension in the elderly by modulating these pathways. ConclusionsYSJ effectively treats senile hypertension. By analyzing the correlation between therapeutic targets and pathways, YSJ's anti-hypertensive effect was achieved by inhibiting lipid peroxidation and matrix degeneration. Combining metabolomics and proteomics provides an effective method for uncovering YSJ's anti-hypertensive mechanisms.

A-088 Clinically Significant Errors Using the Diazyme Total Bile Acid Assay on the Roche c502 Analyzer: Investigating a Result Error Identifies a Novel Large-Scale Testing Error Caused by Reagent Carryover

Abstract Background Thoroughly investigating single patient result errors may identify systemic, large-scale testing errors. The laboratory observed an event where a revision to a Total Bile Acid (TBA, Diazyme Laboratories, Inc.) patient result following a 22S quality control (QC) failure displayed a larger than expected difference (52 to 5mcmol/L). TBA is primarily used for diagnosis and monitoring of intrahepatic cholestasis of pregnancy with results &amp;gt;10mcmol/L considered elevated. Retrospective review of result revisions following QC failures (3/15/2017-5/30/2023) revealed that 50%(20/40) yielded result differences of &amp;gt;10mcmol/L(&amp;gt;20SD based on assay imprecision). The aim of this study was to apply a systematic approach to i) estimate the rate of TBA errors, ii) ensure accurate result reporting during investigation period, iii) perform root cause analysis (RCA), and iv) determine corrective and preventative action(s). Methods Residual samples from TBA testing (6/24-7/5/2023) were retested(n=158), differences &amp;gt;+/-3.0mcmol/L or 15% were confirmed on an alternate analyzer, and reports were revised. Automated repeat testing of TBA samples &amp;gt;10mcmol/L was operationalized 7/6/2023. Initial and repeat TBA results were compared(n=448) and results differing by &amp;gt;+/-20% were remeasured on an alternate analyzer. RCA was conducted using a fishbone diagram. NaOH reagent probe washes were implemented for TBA and the error rate was re-assessed. Assays run prior to TBA samples with errors (n=15) were identified. To assess reagent carryover a residual serum pool (TBA ∼5mcmol/L) was aliquoted into 5 tubes in a sample rack. The first sample in the rack was programmed to run an assay suspected of causing carryover followed by 4 TBA measurements. Mean±standard deviation(SD) TBA concentrations were calculated. TBA was also measured in the liquid reagent for amylase, lipase, acetylcholinesterase (ACE) and fructosamine. Results Initial TBA retesting yielded no errors when initial TBA was ≤10mcmol/L(n=51). For samples with TBA &amp;gt;10mcmol/L(n=107), 9(8.4%) had differences exceeding criteria with 8/9 being revised to ≤10mcmol/L. Analysis of automated patient repeat data showed a 3.8%(17/448) error rate when initial TBA &amp;gt;10mcmol/L. After NaOH reagent probe washes were implemented, the error rate decreased to 0.7%(3/448). Assays run directly before an erroneously high TBA result included: lactate, fructosamine, soluble transferrin receptor(STFR), lipase, and ACE. A serum TBA pool(mean±SD=5.1±0.4 mcmol/L,n=15) measured 37.6±1.2mcmol/L(n=3) and 6.7±0.7mcmol/L(n=3) after lipase and fructosamine, respectively. No other assays demonstrated carryover. TBA in lipase reagent compartment B and C was 653 and 649mcmol/L, respectively, and 653mcmol/L in fructosamine reagent compartment B. Conclusions A single patient TBA result revision was investigated and led to identification of reagent carryover causing erroneously high TBA results on the Roche c502 analyzer. Automatic, real-time, repeat testing was implemented to prevent reporting incorrect patient results until RCA could identify the cause. Occurrence rate of erroneous high TBA results decreased from 3.8% to 0.7% after implementation of NaOH washes on the instruments. Carryover experiments confirmed that lipase and fructosamine assays cause carryover when run prior to a TBA sample due to measurable TBA in the reagent. The lab is pursuing moving the fructosamine and lipase reagent to the Roche Cobas c701 to prevent the issue.