Taurocholic acid is an active promoting factor, not just a biomarker of progression of liver cirrhosis: evidence from a human metabolomic study and in vitro experiments.

BACKGROUND: Previous studies have indicated that bile acid is associated with progression of liver cirrhosis. However, the particular role of specific bile acid in the development of liver cirrhosis is not definite. The present study aims to identify the specific bile acid and explore its possible mechanisms in promoting liver cirrhosis. METHODS: Thirty two cirrhotic patients and 27 healthy volunteers were enrolled. Age, gender, Child-Pugh classification and serum of patients and volunteers were collected. Liquid chromatography tandem mass spectrometry (LC-MS) was utilized to determine concentrations of 12 bile acids in serum. Principal component analysis, fold change analysis and heatmap analysis were used to identify the most changed bile acid. And pathway analysis was used to identify the most affected pathway in bile acid metabolism. Spearman rank correlation analysis was employed to assess correlation between concentrations of bile acids and Child-Pugh classification. Hepatic stellate cells (LX-2) were cultured in DMEM. LX-2 cells were also co-cultured with HepG2 cells in the transwell chambers. LX-2 cells were treated with Na+/taurocholate in different concentrations. Western blot was used to evaluate the expression of alpha smooth muscle actin (α-SMA), type I collagen, and Toll-like receptor 4 (TLR4) in LX-2 cells. RESULTS: Concentrations of 12 bile acids in serum of patients and healthy volunteers were determined with LC-MS successively. Principal component analysis, fold change analysis and heatmap analysis identified taurocholic acid (TCA) to be the most changed bile acid. Pathway analysis showed that TCA biosynthesis increased significantly. Spearman rank correlation analysis showed that concentration of TCA in serum of cirrhotic patients was positively associated with Child-Pugh classification. TCA increased the expression of α-SMA, type I collagen, and TLR4 in LX-2 cells. Moreover, the above effect was strengthened when LX-2 cells were co-cultured with HepG2 cells. CONCLUSIONS: Increased TCA concentration in serum of liver cirrhotic patients is mainly due to increased bile acid biosynthesis. TCA is an active promoter of the progression of liver cirrhosis. TCA promoting liver cirrhosis is likely through activating hepatic stellate cells via upregulating TLR4 expression. TCA is a potential therapeutic target for the prevention and treatment of liver cirrhosis.

Aeromicrobium halocynthiae sp. nov., a taurocholic acid-producing bacterium isolated from the marine ascidian Halocynthia roretzi.

A marine bacterium, strain KME 001(T), was isolated from the siphon tissue of a marine ascidian, Halocynthia roretzi, collected off the coast of Gangneung, Korea. Strain KME 001(T) was a Gram-positive, aerobic, non-spore-forming, rod-shaped and non-motile bacterium. Phylogenetic analysis using 16S rRNA gene sequences revealed that strain KME 001(T) clustered with the genus Aeromicrobium and was closely related to Aeromicrobium ginsengisoli, Aeromicrobium erythreum and Aeromicrobium ponti with 97.7, 97.6 and 97.5 % sequence similarities, respectively. The strain was capable of growth at a variety of temperatures (10-42°C) and over a broad pH range (5.0-10.0). NaCl was required for robust growth of the strain. The diagnostic diamino acid of the cell-wall peptidoglycan was ll-diaminopimelic acid. The predominant menaquinone was MK-9(H(4)). The predominant fatty acids were C(18 : 1)ω9c, C(16 : 0) and 10-methyl C(18 : 0). The DNA-DNA hybridization analyses showed that DNA-DNA relatedness values between strain KME 001(T) and its nearest neighbours, A. ginsengisoli KCTC 19207(T), A. erythreum KCCM 41104(T) and A. ponti KACC 20565(T), were 49.6, 57.1 and 63.5 %, respectively. The DNA G+C content of strain KME 001(T) was 75.9mol%. Chemical investigation of the liquid culture medium of strain KME 001(T) led to the isolation of taurocholic acid as a major secondary metabolite. On the basis of phylogenetic and phenotypic data, strain KME 001(T) is classified as representing a novel species of the genus Aeromicrobium, for which the name Aeromicrobium halocynthiae sp. nov. is proposed. The type strain is KME 001(T) (=JCM 15749(T)=KCCM 90079(T)).

Taurine-deficient diet up-regulated cystathionine beta-synthase monoallele in hemizygous cystathionine beta-synthase knockout mice.

Impaired cystathionine beta-synthase (CBS) causes hyperhomocystinuria and hyperhomocysteinemia, both risk factors for cardiovascular diseases. Reduced CBS activity could decrease cysteine and taurine biosyntheses (metabolites of homocysteine degradation) and lead to less taurocholic acid production with a resultant increased cholesterol content. We hypothesized that a deficiency in CBS genetic material and enzyme activity would reduce taurine synthesis, which would lead to an elevated cholesterol concentration. Both sexes of hemizygous C57BL/6J-Cbs(tm1Unc) [CBS (+/-)] and wild-type C57BL/6J mice [CBS (+/+)] were divided into 2 groups. One group of CBS (+/-) and CBS (+/+) mice was fed a cysteine- and taurine-deficient diet for 8 weeks, and the other group was fed a cysteine, taurine, and vitamin B6-deficient diet for 8 weeks. Significantly higher plasma total homocysteine concentrations occurred in the CBS (+/-) mice than their CBS (+/+) cohorts. Female mice of both genotypes had significantly higher plasma total homocysteine concentrations and significantly lower relative CBS mRNA levels than did male mice. During vitamin B(6) deficiency, plasma total homocysteine concentrations were significantly elevated. Three important findings were a differential sex response of CBS mRNA to feeding the vitamin B(6) diet; CBS (+/-) mice had a significantly lower plasma cholesterol concentration, contrary to what was anticipated; and during feeding, the taurine- and cysteine-deficient diet, CBS mRNA levels in CBS (+/-) mice were reduced only 13% rather than the expected 50%. We conclude that the remaining CBS monoallele is up-regulated in mice when fed a taurine-deficient diet to produce additional CBS mRNA.

Enterohepatic circulation in hamsters with an extracorporeal bile duct.

The present study describes a novel technique for investigations of the enterohepatic circulation in the hamster with an extracorporeal bile duct that allows long-term bile collection in the free-moving animal. The animals recovered for 7 days after the operation before the external loop was cut and bile was collected over a period of 78 h. Under these optimal conditions, initial bile flow (651 +/- 89 microliters per 100 g.h-1) and the secretion rates of biliary lipids were several-fold higher than reported in an earlier study using the acute fistula hamster. Biliary cholesterol secretion amounted to 369 +/- 32 nmol per 100 g.h-1, phospholipid secretion was 2.6 +/- 0.3 mumol per 100 g.h-1, and total bile acid secretion was 31.9 +/- 2.2 mumol per 100 g.h-1. A clearcut diurnal rhythm was demonstrated for bile flow and all biliary constituents. After 9 h the depletion of the bile acid pool was complete and cholic acid synthesis derepressed 1.4-fold from a basal rate of 818 nmol per 100 g.h-1, whereas the derepression of chenodeoxycholic acid synthesis was even less pronounced. Biliary cholesterol output increased 2.2-fold, but the phospholipid secretion was constant during the full experiment. It may be concluded that the technique of an extracorporeal bile duct in the free-moving animal allows studies of bile secretion under optimal conditions. Most likely the bile secretion rates given above approach the physiological rates in the hamster.

Identification of bile acid-CoA: amino acid N-acyltransferase in rat kidney.

A novel location of the bile-acid-conjugating enzyme bile acid-CoA:amino acid N-acyltransferase (BAT) has been discovered in the cytosolic fraction of rat kidney. Both taurine and glycine were utilized as substrates. Formation of bile acid N-acyl amidates was verified by h.p.l.c. by comparison with authentic standards and by specific hydrolysis using cholylglycine hydrolase. Immunoblot analysis using a human liver anti-BAT polyclonal antibody indicated that rat kidney BAT has the same molecular mass as rat liver BAT. These findings suggest that the kidney has a role in bile acid metabolism and physiology.

Feedback regulation of bile acid synthesis in the rat by dietary vs. intravenous cholate or taurocholate.

The regulation of bile acid synthesis was studied (i) in intact or colectomized rats receiving cholate or taurocholate as a dietary supplement and (ii) in experiments using chow-fed animals with a graded intravenous or intraduodenal taurocholate infusion. After the 2-week diet period a bile fistula was established and rates of taurocholate, tauromuricholate and taurochenodeoxycholate secretion were quantitated by high-performance liquid chromatography. During the infusion experiments taurocholate production was calculated from the difference in specific activity of [14C]taurocholate between infusate and bile, whereas tauromuricholate and taurochenodeoxycholate synthesis was derived directly from their secretion rates after pool depletion. Both the 0.5% cholate and taurocholate diet suppressed tauromuricholate and taurochenodeoxycholate secretion nearly totally, but only cholate led to a prolonged inhibition taurocholate synthesis. The diets stimulated total bile acid secretion and expanded the total bile acid pool size 2- to 3-fold, but they also prompted a dramatic increase in the biliary secretion of taurodeoxycholate. In contrast, colectomized animals did not secrete taurodeoxycholate following the cholate diet and, despite a comparable increase in bile acid pool size, tauromuricholate and taurochenodeoxycholate secretion was inhibited to a lesser extent. In addition, the rate of bile acid secretion and synthesis was significantly enhanced when compared to that of intact rats. To determine whether taurocholate affected bile acid synthesis directly, the bile acid was infused intravenously or intraduodenally at varying rates up to 300 mumoles per kg per hr for 54 hr, i.e. a rate exceeding normal total bile acid secretion in these acute bile fistula animals nearly 3-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Bile acid metabolism during development: metabolism of taurodeoxycholic acid in human fetal liver.

The metabolism of tauro-[24-14C]deoxycholic acid was studied in microsomal preparations from fetal liver. The livers were obtained at legal abortions performed between week 13 and 24. Taurodeoxycholic acid was efficiently hydroxylated in the 1 beta- and 7 alpha-positions. The hydroxylase activities did not increase with gestational age. A marked variation in extent of hydroxylation was noted between different preparations. The results are discussed in relation to earlier knowledge of fetal and neonatal bile acid metabolism.

A radioimmunoassay for tauro-beta-muricholic acid suitable for use with isolated rat liver cells.

A new radioimmunoassay which can be used to measure the amounts of tauro-beta-muricholic acid produced by isolated rat hepatocytes in vitro is described. Cross reactivities of other bile acids known to be present in rat liver with the antiserum used in the assay were not sufficient to interfere with the measurement of tauro-beta-muricholic acid. Exogenous taurochenodeoxycholic acid was metabolised by isolated rat hepatocytes concurrently with the appearance of tauro-beta-muricholic acid in the cell.

Synthesis of taurocholate by rat fetal liver in organ culture: effects of cortisol in vitro.

Taurocholate production by fetal hepatic organ cultures was measured by radioimmunoassay. Taurocholate production was maximal on day 1 of in vitro incubation, but was demonstrable in organ cultures maintained for periods up to 15 days. Explants obtained from fetuses of 18 gestational days of age produced only 82 pmol taurocholate per milligram dry weight of tissue during the first 24 h of incubation. Explants obtained from fetuses 21 gestational days of age produced 1,043 pmol taurocholate per milligram dry weight. The presence of cortisol (2.0 X 10(-6) M) in the incubation medium increased synthesis of taurocholate by rat fetal liver in which total taurocholate rose 50-fold above control after 120 h of incubation. In increasing concentrations from 2.0 X 10(-9) M to 2.0 X 10(-7) M, cortisol produced an incremental rise in taurocholate. However, additional increases in cortisol dose failed to provide further stimulation, and taurocholate production was inhibited by cortisol concentrations of 2.0 X 10(-5) M. The results provide further validation for the technique of fetal hepatic organ culture. They demonstrate that taurocholate synthesis is increasing rapidly during the final stages of gestation and show that cortisol augments taurocholate synthesis in a dose-response pattern.

[Taurocholic acid synthesis in dogs maintained on a high cholesterol diet]. / Sintez taurokholevoi kisloty pri soderzhanii sobak na giperkholesterinovoi diete.

Labeled 35S-taurocholic acid synthesis was studied in dogs with gall bladder fistulae, fed high cholesterol diet. During the first period (1--2 months) dietary cholesterol caused a significant increase in the 35S-taurocholic acid synthesis. In 5--6 months, at the period of development of the pathological process in the liver, there was a decrease of synthesis and secretion of bile acids, and of cholesterol sediment in the bile. The level of plasma cholesterol in these dogs increased only slightly.

Kinetic studies on the enzymes conjugating bile acids with taurine and glycine in bovine liver.

Synthesis of glyco- or tauro-cholate from choloyl-CoA and the respective amino acid is shown to be catalysed by the soluble fraction of liver cells. Kinetic evidence supports the conclusion that there are separate enzymes for the synthesis of glycocholate and taurocholate. The kinetic parameters of these enzymes were determined.

Influence of hepatic taurine concentration on bile acid conjugation with taurine.

In vitro, addition of taurine to liver homogenates increases the proportion of cholic acid conjugated with taurine. In the present study, the relation between hepatic taurine concentration and the proportion of infused sodium cholate conjugated with taurine was studied in the whole organ. The isolated perfused liver was studied to eliminate possible transfer of taurine to or from the large extrahepatic poosl present in vivo. During cholate infusion, the proportion of taurocholate excreted in bile decreased, and the proportion of glycocholate increased in a complementary fashion. Infusion of taurine with cholate prevented these changes. Hepatic taurine concentration, calculated from measured hepatic taurine concentrations before and at the end of cholate infusion, fell. Fall in proportion of total bile acid excreted as taurocholate was most rapid at low hepatic taurine concentrations between about 1.4 and 0.65 mumol/g liver. Hepatic taurine concentrations is a major determinant of the proportion of bile acid conjugated with taurine.

Bile acid conjugation in fetal hepatic organ cultures.

A new technique has been developed in which mammalian fetal liver can be maintained in organ culture for prolonged periods with intact structure and function. Near-term rat fetal liver explants were incubated in vitro for periods of up to 3 wk with preservation of normal cellular morphology and intercellular (organ) relationships. [14C]cholate was incorporated into tissue and medium conjugates at a constant rate during 21 days in vitro. During a 24-h incubation with radioactively labeled cholic acid, bile acid conjugates accumulated in tissues to a maximum value by 6 h and maintained this value through 24 h. During the same 24-h incubation with [14C]cholate, conjugates were secreted into the medium at a constant rate. Addition of 8 X 10(-4) M taurine to the medium during a 4-day incubation produced a threefold enhancement in the rate of conjugate formation in tissues and medium. Enhanced conjugation in the presence of additional taurine was due almost entirely to increased taurocholate formation and no significant difference was observed in the amount of glycocholate formed. Exposure of explants to 3.6 X 10(-4) M cycloheximide for prolonged periods resulted in inhibition of conjugate formation, but when this concentration of cycloheximide was maintained for only 24 h a significantly (P less than 0.001) increased rate of conjugate formation was observed. The results indicate that metabolic processes in the organ-culture system are in a state of dynamic equilibrium and that morphologic integrity and specific hepatocytic function are maintained after 21 days in vitro. Preferential taurocholate formation was demonstrated in rat fetal liver, and the data suggest that glycine and taurine interact with separate enzymatic systems in bile acid conjugation. The possible mechanisms that mediate the effect of cycloheximide are discussed.

An enzymatic derivative double isotope assay for measuring tissue levels of taurine.

The development of a new assay for taurine is described. The procedure utilizes the formation of taurocholic acid by rat liver microsomes and is dependent upon the dilution of the specific activity of radioactive taurine by the amount of taurine present in perchloric acid homogenates of tissues. Known amounts of taurine (21/2-300 nmol) are added to incubation vessels to establish a standard curve so that unknown quantities of taurine present in tissue homogenates can be calculated by a graphical comparison. The possible interference by other compounds present in tissue extracts and their removal by passage through columns containing both cation and anion exchange resins is discussed. Conditions are established for the quantitative recovery of taurine after passage through the exchange resins. Measurement of the taurine levels of several rat tissues is presented along with duplicate assays performed on the amino acid analyzer for comparison purposes.

Regulation of bile salt pool size in man.

When the enterohepatic circulation is intact the size of the bile salt pool is largely determined by the frequency of its enterohepatic circulation. This hypothesis, derived from studies of cholate kinetics in coeliac, cholecystectomy, and gall stone patients, represents the simplest interpretation of the data. A corollary is that daily cholate secretion is likely to be normal in these conditions and that therefore the propensity of bile to form cholesterol gall stones is not likely to be directly related to bile salt pool size.

The effect of coeliac disease upon bile salts.

The size and composition of the bile salt pool has been measured in patients with untreated coeliac disease and in control subjects. The total bile salt pool was markedly increased in coeliac patients, the average being 9.2 grams compared with 3.1 grams in controls. Taurocholate synthesis was normal, consistent with its enlarged pool and prolonged half-life. Half-life and pool size were significantly correlated. The composition of the bile salt pool was virtually identical in the two groups. Our findings suggest that as the enterohepatic circulation is slowed by gallbladder inertia, so hepatic surveillance of pool size is diminished.