A validated surrogate analyte UPLC-MS/MS assay for quantitation of TUDCA, TCDCA, UDCA and CDCA in rat plasma: Application in a pharmacokinetic study of cultured bear bile powder.

Bear bile is a valuable medicinal material used in traditional Chinese medicine for over 2000 years. However, developing a substitute has become necessary because of protection measures for this endangered species. The ingredients of in vitro cultured bear bile powder (CBBP) include tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA), ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA, and it has pharmacological properties that are similar to those of natural bear bile powder (NBBP). In this study, the pharmacokinetic parameters of both CBBP and NBBP were measured in rats with a new surrogate analyte LC-MS method using stable isotopes as surrogate analytes (D4-TUDCA, D4-TCDCA, D4-UDCA and D4-CDCA) with response factors validated in authentic matrix (plasma) for simultaneously monitoring the authentic analytes (TUDCA, TCDCA, UDCA and CDCA). The method validation was satisfactory for the linear regression (r, 0.9975-0.9994), precision (RSD intra-day, 0.72-9.35%; inter-day, 3.82-9.02%), accuracy (RE, -12.42-5.67%) and matrix effect (95.53-99.80%), along with analyte recovery (95.90-98.82%) and stability (89.48-101.81%) of surrogate analytes, and precision (RSD intra-day, 1.06- 11.51%; inter-day, 2.23- 11.38%), accuracy (RE, -7.40-10.76%) and stability (87.37-111.70%) of authentic analytes. We successfully applied this method to evaluate the pharmacokinetics of CBBP and NBBP in rats, which revealed the critical in vivo properties of both bear bile preparations.

Deoxycholic Acid, a Metabolite of Circulating Bile Acids, and Coronary Artery Vascular Calcification in CKD.

BACKGROUND: Vascular calcification is common among patients with chronic kidney disease (CKD), and it is associated with all-cause and cardiovascular disease mortality. Deoxycholic acid, a metabolite of circulating bile acids, is elevated in CKD and induces vascular mineralization and osteogenic differentiation in animal models. STUDY DESIGN: Cohort analysis of clinical trial participants. SETTING & PARTICIPANTS: 112 patients with moderate to severe CKD (estimated glomerular filtration rate, 20-45mL/min/1.73m2) who participated in a randomized controlled study to examine the effects of phosphate binders on vascular calcification. PREDICTOR: Serum deoxycholic acid concentration. OUTCOMES: Baseline coronary artery calcification (CAC) volume score and bone mineral density (BMD) and change in CAC volume score and BMD after 9 months. MEASUREMENTS: Deoxycholic acid was assayed in stored baseline serum samples using liquid chromatography-tandem mass spectrometry, CAC was measured using a GE-Imitron C150 scanner, and BMD was determined using computed tomographic scans of the abdomen with calibrated phantom of known density. RESULTS: Higher serum deoxycholic acid concentrations were significantly correlated with greater baseline CAC volume and lower baseline BMD. After adjusting for demographics, coexisting illness, body mass index, estimated glomerular filtration rate, and concentrations of circulating markers of mineral metabolism, including serum calcium, phosphorus, vitamin D, parathyroid hormone, and fibroblast growth factor 23, a serum deoxycholic acid concentration > 58ng/mL (the median) was positively associated with baseline CAC volume (ß=0.71; 95% CI, 0.26-1.16; P=0.003) and negatively associated with baseline BMD (ß = -20.3; 95% CI, -1.5 to -39.1; P=0.04). Serum deoxycholic acid concentration > 58ng/mL was not significantly associated with change in CAC volume score after 9 months (ß=0.06; 95% CI, -0.09 to 0.21; P=0.4). The analysis for the relationship between baseline deoxycholic acid concentrations and change in BMD after 9 months was not statistically significant, but was underpowered. LIMITATIONS: The use of nonfasting serum samples is a limitation because deoxycholic acid concentrations may vary based on time of day and dietary intake. Few trial participants with complete data to evaluate the change in CAC volume score (n=75) and BMD (n=59). No data for changes in deoxycholic acid concentrations over time. CONCLUSIONS: Among patients with moderate to severe CKD, higher serum deoxycholic acid concentrations were independently associated with greater baseline CAC volume score and lower baseline BMD.

Metabolic effects of Lactobacillus reuteri DSM 17938 in people with type 2 diabetes: A randomized controlled trial.

AIMS: To investigate the metabolic effects of 12-week oral supplementation with Lactobacillus reuteri DSM 17938 in patients with type 2 diabetes on insulin therapy. MATERIALS AND METHODS: In a double-blind trial, we randomized 46 people with type 2 diabetes to placebo or a low (108 CFU/d) or high dose (1010 CFU/d) of L. reuteri DSM 17938 for 12 weeks. The primary endpoint was the effect of supplementation on glycated haemoglobin (HbA1c). Secondary endpoints were insulin sensitivity (assessed by glucose clamp), liver fat content, body composition, body fat distribution, faecal microbiota composition and serum bile acids. RESULTS: Supplementation with L. reuteri DSM 17938 for 12 weeks did not affect HbA1c, liver steatosis, adiposity or microbiota composition. Participants who received the highest dose of L. reuteri exhibited increases in insulin sensitivity index (ISI) and serum levels of the secondary bile acid deoxycholic acid (DCA) compared with baseline, but these differences were not significant in the between-group analyses. Post hoc analysis showed that participants who responded with increased ISI after L. reuteri supplementation had higher microbial diversity at baseline, and increased serum levels of DCA after supplementation. In addition, increases in DCA levels correlated with improvement in insulin sensitivity in the probiotic recipients. CONCLUSIONS: Intake of L. reuteri DSM 17938 for 12 weeks did not affect HbA1c in people with type 2 diabetes on insulin therapy; however, L. reuteri improved insulin sensitivity in a subset of participants and we propose that high diversity of the gut microbiota at baseline may be important.

Long-Term Ursodeoxycholic Acid Therapy Does Not Alter Lithocholic Acid Levels in Patients with Cystic Fibrosis with Associated Liver Disease.

OBJECTIVE: To evaluate the fasting and postprandial serum bile acid composition in patients with cystic fibrosis-associated liver disease (CFLD) after chronic administration of ursodeoxycholic acid (UDCA) (20 mg/kg/day). The aim was to specifically focus on the extent of biotransformation of UDCA to its hepatotoxic metabolite, lithocholic acid, because of recent concerns regarding the safety of long-term, high-dose UDCA treatment for CFLD. STUDY DESIGN: Twenty patients with CFLD (median age 16 years, range: 2.4-35.0) prescribed UDCA therapy for at least 2 years were studied. Total and individual serum bile acids were measured by stable-isotope dilution mass spectrometry, in fasting and 2-hour postprandial samples taken during chronic UDCA (20 mg/kg/day) administration. RESULTS: During chronic UDCA administration (median duration 8 years, IQR: 6-16), UDCA became the predominant serum bile acid in all patients (median, IQR: 3.17, 1.25-5.56 µmol/L) and chenodeoxycholic acid concentrations were greater than cholic acid (1.86, 1.00-4.70 µmol/L vs 0.40, 0.24-2.71 µmol/L). The secondary bile acids, deoxycholate and lithocholate, were present in very low concentrations in fasted serum (<0.05 µmol/L). After UDCA administration, 2-hour postprandial concentrations of both UDCA and chenodeoxycholic acid significantly increased (P < .01), but no significant changes in serum lithocholic acid concentrations were observed. CONCLUSION: These data do not support recent suggestions that enhanced biotransformation of UDCA to the hepatotoxic secondary bile acid lithocholic occurs when patients with CFLD are treated with relatively high doses of UDCA.

Prevention effect of orally active heparin conjugate on cancer-associated thrombosis.

Thrombogenesis is a major cause of morbidity and mortality in cancer patients. Prophylaxis with low-molecular-weight heparin (LMWH) is recommended for cancer patients, but requires non-parenteral delivery methods for long-term treatments. In this study, we sought to generate a new oligomeric-bile acid conjugate of LMWH that can be used for oral delivery. We first synthesized a tetramer of deoxycholic acid (tetraDOCA), which was site-specifically conjugated at the end saccharide of LMWH. When LMWH-tetraDOCA conjugate (LHe-tetraD) was orally administered at a dose of 5 mg/kg in ICR mice, the maximum anti-factor Xa level was increased up to 0.62±0.05 IU/mL without any evidence of liver toxicity, gastrointestinal damage, or thrombocytopenia. The cancer-associated thrombosis was induced in tumor-bearing mice by local heat application, and the fibrin deposition in tumors was evaluated. The oral administration of LHe-tetraD (either a single dose or multiple daily doses for up to 10 days) in mice substantially abolished the coagulation-dependent tropism of fibrinogen in the heated tumors and significantly decreased hemorrhage, compared to the mice treated with saline or subcutaneous injection of LMWH. Thus, the anticoagulation effect of oral LHe-tetraD invokes the benefits of oral delivery and promises to provide an effective and convenient treatment for cancer patients at risk of thrombosis.

Experimental central nervous system aspergillosis therapy: efficacy, drug levels and localization, immunohistopathology, and toxicity.

We have shown previously that high-dose lipid amphotericin preparations are not more efficacious than lower doses in aspergillosis. We studied toxicity, drug concentrations and localization, and quantitative infection concurrently, using a 4-day model of central nervous system (CNS) aspergillosis to assess early events. Mice given Aspergillus fumigatus conidia intracerebrally, under a cyclophosphamide immunosuppressive regimen, were treated for 3 days (AmBisome at 3 or 10 mg/kg of body weight, Abelcet at 10 mg/kg, amphotericin B deoxycholate at 1 mg/kg, caspofungin at 5 mg/kg, or voriconazole at 40 mg/kg). Sampling 24 h after the last treatment showed that AmBisome at 3 but not at 10 mg/kg, as well as Abelcet, caspofungin, and voriconazole, reduced brain CFU. All regimens reduced renal infection. Minor renal tubular changes occurred with AmBisome or Abelcet therapy, whereas heart, lung, and brain showed no drug toxicity. Amphotericin B tissue and serum concentrations did not correlate with efficacy. Endothelial cell activation (ICAM-1 and P-selectin in cerebral capillaries) occurred during infection. Amphotericin B derived from AmBisome and Abelcet localized in activated endothelium and from Abelcet in intravascular monocytes. In 10-day studies dosing uninfected mice, minor renal tubular changes occurred after AmBisome or Abelcet at 1, 5, or 10 mg/kg with or without cyclophosphamide treatment; nephrosis occurred only with Abelcet in cyclophosphamide-treated mice. Hepatotoxicity occurred with AmBisome and Abelcet but was reduced in cyclophosphamide-treated mice. Marked CFU reduction by AmBisome at 3 mg/kg occurred in association with relatively more intense inflammation. Abelcet renal localization appears to be a precursor to late nephrotoxicity. Hepatotoxicity may contribute to high-dose Abelcet and AmBisome failures. Our novel observation of endothelial amphotericin localization during infection may contribute to amphotericin mechanism of efficacy.

Bile acid metabolism in cirrhosis. IV. Characterization of the abnormality in deoxycholic acid metabolism.

Several recent studies have demonstrated that patients with cirrhosis frequently lack deoxycholic acid in bile and plasma. In order to explain this observation, comparative experiments on the colonic absorption of deoxycholic acid and on the colonic conversion of cholic to deoxycholic acid were carried out in the cirrhotic patients with normal and very low percentages of deoxycholic acid. Deoxycholic or cholic acid (100 mg) plus 5 muc of each [14C] bile acid were administered by enema to 8 patients with and 5 without liver disease. Deoxycholic acid produced a significant increase in the percentage of biliary deoxycholic acid in patients with cirrhosis. However, the rate of appearance of 14C-deoxycholic acid in patients with cirrhosis was slower than in normal control subjects. Distribution of the 14C activity among the bile acids indicated that rehydroxylation of deoxycholic to cholic acid did not occur. The distribution of 14C activity in biliary bile acids after the rectal administration of [14C]cholic acid showed that patients with severe cirrhosis converted [14C]cholic to [14C]deoxycholic acid at a much slower rate than did cirrhotic patients with normal percentages of biliary deoxycholic acid. Feeding of cholic acid (450 mg per day) for 3 days to 4 cirrhotic patients resulted in a 2-fold increase in the percentage of biliary cholic acid, but only a small increase in the percentage of deoxycholic acid. In a separate group of 9 cirrhotic patients, fecal bile acid analysis indicated that cirrhotic patients had a significantly lower percentage of deoxycholic acid than 12 patients without liver disease; there was no significant difference in fecal lithocholic acid. The data suggest that alteration of bacterial flora and/or altered conditions for bacterial 7alpha-dehydroxylase enzyme activity in the colon could account for the virtual absence of biliary deoxycholic acid in severely cirrhotic patients.