Oral gavage of nano-encapsulated conjugated acrylic acid-bile acid formulation in type 1 diabetes altered pharmacological profile of bile acids, and improved glycaemia and suppressed inflammation.

BACKGROUND: Ursodeoxycholic acid (UDCA) is a secondary hydrophilic bile acid, metabolised in the gut, by microbiota. UDCA is currently prescribed for primary biliary cirrhosis, and of recently has shown ß-cell protective effects, which suggests potential antidiabetic effects. Thus, this study aimed to design targeted-delivery microcapsules for oral uptake of UDCA and test its effects in type 1 diabetes (T1D). METHODS: UDCA microcapsules were produced using alginate-NM30 matrix. Three equal groups of mice (6-7 mice per group) were gavaged daily UDCA powder, empty microcapsules and UDCA microcapsules for 7 days, then T1D was induced by alloxan injection and treatments continued until mice had to be euthanised due to weight loss > 10% or severe symptoms develop. Plasma, tissues, and faeces were collected and analysed for bile acids' concentrations. RESULTS: UDCA microcapsules brought about reduction in elevated blood glucose, reduced inflammation and altered concentrations of the primary bile acid chenodeoxycholic acid and the secondary bile acid lithocholic acid, without affecting survival rate of mice. CONCLUSION: The findings suggest that UDCA exerted direct protective effects on pancreatic ß-cells and this is likely to be associated with alterations of concentrations of primary and secondary bile acids in plasma and tissues. Three equal groups of mice were gavaged daily UDCA (ursodeoxycholic acid) powder, empty microcapsules and UDCA microcapsules for 7 days, then T1D was induced and treatments continued until mice had to be euthanised. UDCA microcapsules brought about reduction in elevated blood glucose, reduced inflammation and altered concentrations of the primary bile acid chenodeoxycholic acid and the secondary bile acid lithocholic acid, without affecting survival rate of mice. The findings suggest that UDCA exerted direct protective effects on pancreatic ß-cells and this is likely to be associated with alterations of concentrations of primary and secondary bile acids in plasma and tissues.

Liquid crystals-based sensor for the detection of lithocholic acid coupled with competitive host-guest inclusion.

A simple, low-cost and label-free strategy for detecting lithocholic acid (LCA) was designed at the liquid crystals (LCs)/aqueous interface via competitive host-guest inclusion. In this method, sodium dodecyl sulfate (SDS) was initially adsorbed on the fluid interface and induced LCs to adopt the homeotropic ordering. Inclusion complexation of SDS and ß-cyclodextrin (ß-CD) disturbed interaction between LCs and SDS and evoked LCs to keep a tilted alignment. When injecting LCA into the mixed solution of SDS and ß-CD, SDS excluded from the cavity of ß-CD by competitive host-guest inclusion and could be re-adsorbed at the LCs/aqueous interface, resulting in the orientational transition of LCs from tilted to homeotropic state. Correspondingly, a bright-to-dark optical response was observed under polarized optical microscope (POM). The as-prepared LCs-based sensor could detect LCA as low as about 2 µM in aqueous solution. Moreover, the practicability of the approach was validated by monitoring the known amount of LCA in human urine. This work offers an appealing approach for the detection of LCA which has a great potentiality in clinical diagnosis.

Role of glucuronidation for hepatic detoxification and urinary elimination of toxic bile acids during biliary obstruction.

Biliary obstruction, a severe cholestatic condition, results in a huge accumulation of toxic bile acids (BA) in the liver. Glucuronidation, a conjugation reaction, is thought to protect the liver by both reducing hepatic BA toxicity and increasing their urinary elimination. The present study evaluates the contribution of each process in the overall BA detoxification by glucuronidation. Glucuronide (G), glycine, taurine conjugates, and unconjugated BAs were quantified in pre- and post-biliary stenting urine samples from 12 patients with biliary obstruction, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The same LC-MS/MS procedure was used to quantify intra- and extracellular BA-G in Hepatoma HepG2 cells. Bile acid-induced toxicity in HepG2 cells was evaluated using MTS reduction, caspase-3 and flow cytometry assays. When compared to post-treatment samples, pre-stenting urines were enriched in glucuronide-, taurine- and glycine-conjugated BAs. Biliary stenting increased the relative BA-G abundance in the urinary BA pool, and reduced the proportion of taurine- and glycine-conjugates. Lithocholic, deoxycholic and chenodeoxycholic acids were the most cytotoxic and pro-apoptotic/necrotic BAs for HepG2 cells. Other species, such as the cholic, hyocholic and hyodeoxycholic acids were nontoxic. All BA-G assayed were less toxic and displayed lower pro-apoptotic/necrotic effects than their unconjugated precursors, even if they were able to penetrate into HepG2 cells. Under severe cholestatic conditions, urinary excretion favors the elimination of amidated BAs, while glucuronidation allows the conversion of cytotoxic BAs into nontoxic derivatives.

Feed-forward regulation of bile acid detoxification by CYP3A4: studies in humanized transgenic mice.

Bile acids are potentially toxic end products of cholesterol metabolism and their concentrations must be tightly regulated. Homeostasis is maintained by both feed-forward regulation and feedback regulation. We used humanized transgenic mice incorporating 13 kb of the 5' regulatory flanking sequence of CYP3A4 linked to a lacZ reporter gene to explore the in vivo relationship between bile acids and physiological adaptive CYP3A gene regulation in acute cholestasis after bile duct ligation (BDL). Male transgenic mice were subjected to BDL or sham surgery prior to sacrifice on days 3, 6, and 10, and others were injected with intraperitoneal lithocholic acid (LCA) or vehicle alone. BDL resulted in marked hepatic activation of the CYP3A4/lacZ transgene in pericentral hepatocytes, with an 80-fold increase in transgene activation by day 10. Individual bile acids were quantified by liquid chromatography/mass spectrometry. Serum 6beta-hydroxylated bile acids were increased following BDL, confirming the physiological relevance of endogenous Cyp3a induction to bile acid detoxification. Although concentrations of conjugated primary bile acids increased after BDL, there was no increase in LCA, a putative PXR ligand, indicating that this cannot be the only endogenous bile acid mediating this protective response. Moreover, in LCA-treated animals, 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside staining showed hepatic activation of the CYP3A4 transgene only on the liver capsular surface, and minimal parenchymal induction, despite significant liver injury. This study demonstrates that CYP3A up-regulation is a significant in vivo adaptive response to cholestasis. However, this up-regulation is not dependent on increases in circulating LCA and the role of other bile acids as regulatory molecules requires further exploration.

Fetal bile acid metabolism: analysis of urinary 3beta-monohydroxy-delta(5) bile acid in preterm infants.

To elucidate the urinary concentration of total bile acids after birth and the profile of the usual and unusual urinary bile acids, especially 3beta-hydroxy-5-cholen-24-oic acid (Delta(5)-3beta-ol), we measured the concentrations of 13 bile acids in the urine from preterm infants vs. full-term controls by gas chromatography-mass spectrometry. The urinary concentration of total bile acids in early preterm infants below 32 weeks of gestational age significantly exceeded that of the late preterm and full-term infants (p < 0.0005). The major urinary bile acids in early preterm infants were cholic acid, 1beta,3alpha,7alpha,12alpha-tetrahydroxy-5beta-cholan-24-oic acid and Delta(5)-3beta-ol. In conclusion, the high urinary concentrations of total bile acids in preterm infants may be due to an overproduction, or more likely to a low hepatic bile acid clearance. An alternative fetal pathway, the acidic pathway, may be a major route of bile acid biosynthesis in preterm infants.

Differences in the metabolism and disposition of ursodeoxycholic acid and of its taurine-conjugated species in patients with primary biliary cirrhosis.

The clinical effectiveness of ursodeoxycholate in the treatment of liver disease may be limited by its poor absorption and extensive biotransformation. Because in vitro and in vivo studies suggest that the more hydrophilic bile acid tauroursodeoxycholate has greater beneficial effects than ursodeoxycholate, we have compared for the first time the absorption, metabolism, and clinical responses to these bile acids in patients with primary biliary cirrhosis (PBC). Twelve female patients with PBC were sequentially administered tauroursodeoxycholate and ursodeoxycholate (750 mg/d for 2 months) in a randomized, cross-over study. Bile acids were measured in serum, duodenal bile, urine, and feces by gas chromatography-mass spectrometry (GC-MS). Biliary ursodeoxycholate enrichment was higher during tauroursodeoxycholate administration (32.6% vs. 29.2% during ursodeoxycholate; P <.05). Lithocholic acid concentration was consistently higher in all biological fluids during ursodeoxycholate administration. Fecal bile acid excretion was the major route of elimination of both bile acids; ursodeoxycholate accounted for 8% and 23% of the total fecal bile acids during tauroursodeoxycholate and ursodeoxycholate administration, respectively (P <.05). Tauroursodeoxycholate was better absorbed than ursodeoxycholate, and, although it was partially deconjugated and reconjugated with glycine, it underwent reduced biotransformation to more hydrophobic metabolites. This comparative study suggests that tauroursodeoxycholate has significant advantages over ursodeoxycholate that may be of benefit for long-term therapy in PBC.

Urinary excretion of lithocholic acid and its conjugates by the bile duct-ligated rat.

The 3-O-glucuronide of lithocholic acid has been shown to be a potent cholestatic agent in rats. However, even after the onset of lithocholic acid glucuronide-induced cholestasis, little of the administered material was recovered in urine. To determine whether this phenomenon was related to the steroid moiety or the form of conjugation, small doses of radiolabeled lithocholic acid glucuronide, lithocholic acid, taurolithocholic acid and/or lithocholic acid sulfate were administered to rats with ligated bile ducts. Urinary excretion of isotope was followed for 24 hr and urinary metabolites of the administered compounds were identified by thin-layer chromatography. Lithocholic and taurolithocholic acids were slowly but relatively efficiently excreted in urine with 73% and 91% of the dose, respectively, recovered in urine over 24 hr. More than 80% of the label in urine from animals receiving these two compounds was in the form of taurine-conjugated beta-muricholic acid. In contrast, lithocholic acid 3-glucuronide and 3-sulfate were poorly excreted: 9% and 12% of the administered doses, respectively, were recovered in urine in 24 hr. Of the small amount of label in urine from rats given the glucuronide, 90% was identified as lithocholic and taurolithocholic acid glucuronides. When lithocholic acid sulfate was given, thin-layer chromatography of urine showed two peaks, which were tentatively identified as tauromurideoxycholic and taurolithocholic acid sulfates. More definitive identification was not possible because of the small amount of the administered dose excreted in urine in these forms.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered bile acid profiles in duodenal bile and urine in diabetic subjects.

The bile acid composition in duodenal bile was analysed in 22 diet-treated and 11 insulin-treated middle-aged patients with diabetes mellitus and in 20 normoglycaemic controls. In 10 subjects with diabetes mellitus the bile acid profile in urine was also investigated. In the non-insulin-dependent diabetic patients the percentage of cholic acid was reduced and that of deoxycholic acid increased. As a highly significant finding there was a three-fold increase of the percentage of 12-ketolithocholic acid in duodenal bile in non-insulin-dependent diabetics, whereas the bile acid composition in insulin-dependent diabetics was similar to that in a control group. The percentage of 12-ketolithocholic acid in duodenal bile was positively correlated to the percentage in urine. In nine of the subjects studied, 12-ketolithocholic acid was the major individual bile acid in urine. It constituted 36.3 +/- 4.4% of the bile acids analysed and the excretion was 6.1 +/- 2.3 mumol 24 h-1. Together with 3 alpha, 12 beta-dihydroxy-5 beta-cholanoic acid it was predominantly present in the glycine conjugate fraction, whereas in bile its conjugation was similar to that of the other bile acids. The results may reflect an increased formation of secondary bile acids from cholic acid combined with a metabolic disturbance in non-insulin-dependent diabetics affecting the oxidoreduction of bile acids at C-12.

Urinary bile acid excretion in correlation to liver histopathology in cystic fibrosis.

Urinary bile acid excretion and liver morphology were compared in 25 patients with cystic fibrosis (CF). None showed clinical signs of liver disease. Most of the patients had normal liver function tests. Bile acids were determined in 24-h samples by a modification of the method of Almé. All patients had increased urinary excretion of trihydroxy bile acids, mainly cholic, 3 beta, 7 beta, 12 alpha- and 3 alpha, 7 beta, 12 alpha-trihydroxy-5 beta-cholanoic acids. Lithocholic acid excretion was lower in CF than in normal children. The urinary excretion of 3 beta-hydroxy-5-cholenoic acid was not increased in CF. In three patients with cirrhosis the urinary excretion of chenodeoxycholic acid was increased. The ratio of cholic to 3 beta-hydroxy-5-cholenoic acids was increased in all but three patients, and the ratio of chenodeoxycholic to 3 beta-hydroxy-5-cholenoic acids was increased in those with cirrhosis. These ratios differed more between cirrhotic and non-cirrhotic CF patients in this series than the ratio of cholic to chenodeoxycholic acids.

Synthesis of bile acid monosulphates by the isolated perfused rat kidney.

Perfusion of an isolated rat kidney with labelled bile acids, in a protein-free medium, resulted in the urinary excretion of the labelled bile acid, 3% being converted into polar metabolities in 1h. These metabolities were neither glycine nor taurine conjugates, nor bile acid glucuronides, and on solovolysis yielded the free bile acid. On t.l.c. the metabolite of [24-14C]lithocholic acid had the mobility of lithocholate 3-sulphate. The principal metabolite of [24-14C]chenodeoxycholic acid had the mobility of chenodeoxycholate 7-sulphate; trace amounts appeared as chenodeoxycholate 3-sulphate. [35S]sulphate was incorporated in chenodeoxycholic acid by the kidney, resulting in a similar pattern of sulphation. No disulphate salt of chenodeoxycholic acid was detected. These findings lend support to the hypothesis that renal synthesis may account for some of the bile acid sulphates present in urine in the cholestatic syndrome in man.

Sulfation and renal excretion of bile salts in patients with cirrhosis of the liver.

Renal excretion of bile salts was studied in 17 patients with cirrhosis of the liver. The average quantity of bile salts in urine was 10.2 plus or minus 8.3 mg per 24 hr, 56% of which were sulfated. Of the individual urinary bile salts, 24% oithocholate were sulfated. In contrast, neither sulfated nor nonsulfated bile salts could be detected in urine from 2 normal subjects. Kinetics of bile salt metabolism was measured in 2 of the cirrhotic patients after oral administration of [14C] cholate and [3H] chenodeoxycholate. Approximately 3 to 12% of bile salts synthesized in liver were excreted in urine. Most urinary bile salts (76 to 80%) were sulfated, whereas only 4 to 5% of serum bile salts and 7 to 10% of biliary bile salts were sulfated. Renal clearance of cholate was more than 3 times greater than the clearance of chenodeosycholate or deoxycholate. Renal clearance of sulfated bile salts was 20 to 200 times greaterthan the clearance of the corresponding nonsulfated bile salts.