The diversity of ursodeoxycholic acid precursors from bile waste of commercially available fishes, poultry and livestock in Indonesia

Ursodeoxycholic acid (UDCA), a secondary bile acid (BA), has been used as a drug to treat various liver diseases. UDCA is synthesised from cholic or chenodeoxycholic acid (CA/CDCA), two primary BAs frequently used as the starting materials. Nowadays, swine, cattle, and poultry bile are the main sources of those BAs. However, other commercial animals could be promising sources as well. We identified two livestock, two poultries, and eight fishes that are commercially cultivated in Indonesia. Four free BAs including CA, CDCA, deoxycholic acid (DCA), and lithocholic acid (LA) were identified for their occurrences using thin-layer chromatography and high-performance liquid chromatography. CA was detected in cow, duck, red tilapia, gourami, the common carp, and grouper, whereas CDCA was only detected in two poultries and the common carp. The occurrence of DCA was common and abundant in most tested animals. In contrast, the presence of LA was found to be very low in all samples. The biliary bile of tilapia has been found to contain a high abundance of free CA (43% of the total bile). A simple extraction was able to purify CA from biliary bile of tilapia. This is a new promising and competitive source of CA.

Decreased C-reactive protein induces abnormal vascular structure in a rat model of liver dysfunction induced by bile duct ligation

BACKGROUND/AIMS: Chronic liver disease leads to liver fibrosis, and although the liver does have a certain regenerative capacity, this disease is associated with dysfunction of the liver vessels. C-reactive protein (CRP) is produced in the liver and circulated from there for metabolism. CRP was recently shown to inhibit angiogenesis by inducing endothelial cell dysfunction. The objective of this study was to determine the effect of CRP levels on angiogenesis in a rat model of liver dysfunction induced by bile duct ligation (BDL). METHODS: The diameter of the hepatic vein was analyzed in rat liver tissues using hematoxylin and eosin (H&E) staining. The expression levels of angiogenic factors, albumin, and CRP were analyzed by real-time PCR and Western blotting. A tube formation assay was performed to confirm the effect of CRP on angiogenesis in human umbilical vein endothelial cells (HUVECs) treated with lithocholic acid (LCA) and siRNA-CRP. RESULTS: The diameter of the hepatic portal vein increased significantly with the progression of cirrhosis. The expression levels of angiogenic factors were increased in the cirrhotic liver. In contrast, the expression levels of albumin and CRP were significantly lower in the liver tissue obtained from the BDL rat model than in the normal liver. The CRP level was correlated with the expression of albumin in hepatocytes treated with LCA and siRNA-CRP. Tube formation was significantly decreased in HUVECs when they were treated with LCA or a combination of LCA and siRNA-CRP. CONCLUSION: CRP seems to be involved in the abnormal formation of vessels in hepatic disease, and so it could be a useful diagnostic marker for hepatic disease.

Secondary bile acids effects in colon pathology. Experimental mice study

ABSTRACTPURPOSE:To assess whether deoxycholic acid (DOC) and lithocholic acid (LCA) administered in a period of six months in a concentration of 0.25% may have a carcinogenic role in mice colon.METHODS:The study used C57BL6 female mice divided into four groups. The control group received a balanced diet and the others received diets supplemented with 0.25% DOC, 0.25% LCA and 0.125% DOC+0.125% LCA, respectively. After euthanasia, the lesions found in the resected gastrointestinal tracts were stained with hematoxylin-eosin and examined microscopically.RESULTS:No gastrointestinal tract changes were observed in the control group, while hyperplastic Peyer's patches in the small intestine, flat adenomas with mild dysplasia and chronic colitis at the level of the colon were found in all three test groups. The colonic lesions prevailed in the proximal colon. The highest number of flat adenoma lesions (8), hyperplasia of Peyer's patches (25) and chronic colitis (2) were found in mice fed with diet and LCA.CONCLUSION: Precancerous or cancerous pathological lesions could not be identified. Instead, adenomatous colonic injuries occurred in a shorter period of time (six months), compared to the reported data.

Metabolomic Study of a Diagnostic Model for the Metabolites of Stool Fat / 대한소화기학회지

BACKGROUND/AIMS: Metabolomics is a powerful tool for measuring low-molecular-weight metabolites in an organism at a specified time under specific environmental conditions. The aim of this study was to determine the usefulness of metabolomics in identifying the metabolites in stool-fat-positive specimens, and to establish whether the results could be used to predict the long-term prognosis. METHODS: Fecal specimens were collected from 52 subjects with bowel habit change. The subjects were accessed using Rome III questionnaires and Bristol stool scale form, and followed after three years. The feces samples were centrifuged and the resulting extracts reconstituted for liquid chromatography/mass spectrometry analysis. The datasets were autoscaled, log-transformed, and mean-centered in a column-wise fashion prior to principal-components analysis and partial least-squares-discrimination analysis modeling. RESULTS: Fecal samples from 10 of the 52 patients gave a positive stool-fat result of 30-100 microm; those of the remaining 42 contained neither fatty acids nor neutral fats. The peak intensities of lithocholic acid (p=0.001), lysophosphatidyl ethanolamine (lysoPE) 16:0 (p=0.015), and lysoPE 18:1/0:0 (p=0.014) were correlated with the size of the fatty acid. Subjects with positive stool-fat result showed higher score in Bristol stool scale form than those with negative stool-fat result at initial (p=0.040) and after three years (p=0.012). CONCLUSIONS: The metabolomic assay of stool fatty acid revealed mainly lysoPEs and lithocholic acid. The size of the fatty acid was correlated with higher concentrations of lysoPEs and lithocholic acid in stool-fat-test-positive specimens and related to loose stool even after three years of follow-up period.

Alterações hepatobiliares induzidas pelos ácidos cólico e litocólico: estudo experimental em hamsters / Hepatobiliaries alterations induced by cholic and lithocholic acids: hamsters experimental study

A atresia de vias biliares é a causa mais comum de icterícia obstrutiva, cirrose e transplante hepático da infância. Sua etiopatogenia permanente desconhecida. Dentre várias teorias, uma propõe que a enfermidade pode ser causada pelo efeito tóxico de ácidos biliares monohidroxilados no sistema hepatobiliar fetal e neonatal. As características do metabolismo biliar nesta fase da vida e possíveis alterações bioquímicas desses ácidos poderiam causar reaçäo inflamatória e obstruçäo ductal. Ainda näo foi feito qualquer estudo experimental da açäo desses ácidos sobre o sistema hepatobiliar durante a gravidez. Neste trabalho, avaliaram-se os efeitos tóxicos provocados pela ingestäo de um ácido biliar trihidroxilado, o cólico, e um monohidroxilado, o litocólico, sobre o sistema hepatobiliar de hamsters durante os períodos gestacional e perinatal. A escolha deste animal deve-se à semelhança de seu metabolismo biliar com o humano. A ingestäo de ácido litocólico a 0,5 por cento durante a gestaçäo de hamsters, provocou proliferaçäo ductal/ductular acentuada, sinais inflamatórios, degeneraçäo e regeneraçäo celular hepática, hiperplasia do epitélio dos ductos extra-hepáticos maternos e aborto. Tanto o ácido cólico a 0,5 por cento como o ácido litocólico a 0,25 por cento, quando ingeridos por hamsters grávidas, provocaram proliferaçäo ductal/ductular e lesäo inflamatória hepatobiliar em graus variáveis no animal adulto e de leve intensidade nos filhotes. Induziu, ainda, a reduçäo da ninhada. Portanto, verificou-se que a ingestäo destes ácidos biliares por hamsters durante o período gestacional provocou toxicidade variável sobre o sistema hepatobibliar materno e de recém-nascidos

The relationship of bacterial and Helicobacter infection to composition of bile acid in biliary tract diseases / 대한내과학회지

BACKGROUND: Bacterial and Helicobacter gene were commonly detected in diseased human bile, although the meaning of the presence of Helicobacter in biliary tract is still unclear. The aim of this study was to evaluate the changes of bile acid composition in bacterial and Helicobacter infected bile, and to determine whether Helicobacter pylori might grow in human bile or not. METHODS: Thirty bile samples were obtained by percutaneous transhepatic biliary drainage or gallbladder puncture during cholecystectomy. According to the polymerase chain reaction analysis using bacterial 16S rRNA and Helicobacter genus specific 16S rRNA primers, 3 groups were divided; Group I; no presence of any bacterial DNA, Group II; positive bacterial DNA only, Group III; positive bacterial and Helicobacter DNA. Bile acid analysis for deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA) was performed by high performance liquid chromatography. And then Helicobacter pylori was tried to culture in broth mixed with human bile at a final bile concentration of 50%. RESULTS: The concentrations of DCA in group II and III were very low and significantly reduced compared to group I (p<0.01, respectively). The concentrations of LCA or UDCA were not shown any relationships between groups. Helicobacter pylori has grown actively in the broth mixed with human bile containing both of less than 0.1 gm/L of DCA and CDCA, compared to no growth in media mixed with human bile containing more than 3.0 gm/L of DCA and/or CDCA. CONCLUSION: DCA seems to have the strongest antibacterial effect. Helicobacter pylori is likely to grow in human bile containg very low concentrations of CDCA and DCA.

Gastric Stump Cancer

PURPOSE: Gastric stump cancer is defined as a cancer that develops in the stomach after a resection in cases of non-malignant or malignant gastric disease. The interval between the gastrectomy and the detection of gastric stump cancer must be over 5 years. Since duodenogastric reflux gastritis is a precancerous condition and one of the most important factors inducing gastric stump cancer, we compared the bile-acid content of gastric juice between gastric stump cancer patients and controls. MATENRIALS AND METHODS: To evaluate retrospectively the surgical treatment of patients with gastric stump cancer, we reviewed the cases histories of 1016 stomach cancer patients who had been operated on at the Department of General Surgery, Kosin University Gospel Hospital, between 1995 and 1998. The gastric juice was collected during the operations on the gastric stump cancer patients by using a needle puncture of the fundus of the stomach and during the endoscopic examinations of the control subjects. The samples were analyzed for various bile acids (gas chromatography/mass spectrometry). RESULTS: The 6 gastric stump cancer cases accounted for 0.6% of all gastric cancer patients; 5 patients were first operated on for a peptic ulcer and the remaining one for an adenocarcinoma of the stomach. All of the cases were men. The reconstruction method after the initial gastrectomy was a Billroth II in all cases. The sites of the gastric stump cancer were the anastomotic sitein 2 patients, the upper body in 2, the fundus in 1 and the cardia in 1. The operative methods were 3 total gastrectomies, 2 subtotal gastrectomies with Roux en Y anastomosis, and 1 partial gastrectomy with lymph node dissection and had a curative intention in all patients. All of the patients were still surviving at the time of this report. The gastric juices of 4 gastric stump patients showed significantly higher contents of cholic acid (36.42microgram/ ml) compared to the gastric juices of 35 control subjects (12.82microgram/ml)(p< or =0.0001). Chenodeoxycholic acid and lithocholic acid were not significantly different. CONCLUSION: The gastric juice of gastric stump cancer patients contained a significantly higher cholic acid content. At the time of the initial gastrectomy, an operative method that prevents duodenogastric reflux may prevent or minimize the development of gastric stump cancer, and more aggressive surgical treatment may improve survival.

Toxicity of Bile Acids on Colon Cancer Cell Lines / 대한암학회지

PURPOSE: Cytotoxicity of the bile acids on colon cancer cell lines was studied to know which bile acid was most cytotoxic to colonic mucosal epithelium. We performed agarose gel electrophoresis whether this toxicity was caused by detergent effect of the bile acids or by apoptotic pathway. MATERIALS AND METHODS: HT29, LoVo, SW620 colon cancer cell lines were exposed to lithocholate, cholate, deoxycholate and chenodeoxycholate with 50, 100, 150, 200, 250, 300 pM as final concentration in DMEM culture media for short time (for 2 hours) and for long time (for 5 days). Agarose gel electrophoresis was performed on each colon cancer cell lines (HT29, LoVo, SW620, SW480) after 1, 2, 3, 4, 5 days exposure to deoxycholate with 150 pM concentration to detect intemucleosomal fragmentation. RESULTS: There was no toxicity after short time exposure in all bile acids concentration and in all colon cancer cell lines. Of the bile acids, deoxycholate was most toxic for all colon cancer cell lines. And DNA fragmentation was noticed after 2 days exposure with deoxycholate. Only LoVo cell line showed apoptotic DNA pattern after 4 days of exposure with deoxycholate. CONCLUSION: Bile acids (especially deoxycholate) are suggested to be possible agents to cause apoptosis in colonic mucosal epithelium.

Induction of apoptosis by bile acids in HepG2 human hepatocellular carcinoma cells

We studied the effects of bile acids on the induction of apoptosis in HepG2 human hepatocellular carcinoma cells. Treatment with either ursodeoxycholic acid (UDCA) or lithocholic acid (LCA) resulted in a dose- and time-dependent decrease in cell viability assessed by MTT assay. Both UDCA and LCA also induced genomic DNA fragmentation, a hallmark of apoptosis, indicating that the mechanism by which these bile acids induce cell death was through apoptosis. Cycloheximide, a protein synthesis inhibitor, blocked the apoptosis induced by these bile acids, implying that new protein synthesis may be required for the apoptosis. Intracellular Ca2+ release blockers (dantrolene and 3,4,5-trimethoxybenzoic acid-8-(diethylamino)octyl ester) inhibited decreased cell viability and DNA fragmentation induced by these bile acids. Treatment of HepG2 cells with calcium ionophore A23187 induced DNA fragmentation. These results suggest that UDCA and LCA induce apoptosis in the HepG2 cells and that the activation of intracellular Ca2+ signals may play an important role in the apoptosis induced by these bile acids.

Determinacion de los acidos colico y sulfolitoglicocolico en diferentes hepatopatias. / Determination of cholic and sulfolithocholic acids in different hepatopathies

e determino en el suero el acido colico conjugado (acido glicocolico) (GC) y el acido sulfolitocolico conjugado (acido sulfolitoglicocolico) (SLGC) en 35 hepatitis cronicas agresivas (HAC), 22 cirrosis, 11 cirrosis biliares primarias (CBP), 11 prurito del embarazo y en 20 controles normales. El grupo control tuvo un valor promedio para GC de 21 ug% con una dispersion de +/- 14 ug% y para SLGC de 36 ug% con dispersion de +/- 9 ug%. La frecuencia de alteracion de GC y SLGC en las hepatitis cronicas fue baja (14% y 9%) y en la cirrosis (23% y 14%).En el prurito del embarazo se elevo a 64% y 45%. En cambio en las CBP fue significativamente elevada (100% y 90%) (p< 0.001 y p< 0.06). Los valores absolutos en las distintas patologias mostraron valores promedios diferentes pero con muy amplia dispersion que impidieron ver alguna relacion entre dichos valores absolutos y las distintas enfermedades.Expresion de la alteracion del metabolismo de los acidos biliares en la patologia hepatica fue la determinacion postprandial de GC y SLGC en 20 HCA en quienes la frecuencia de valores anormales subiera de 14% y 9% en ayunas a 60% y 45% postprandial.