Bile acid metabolism in cirrhotic liver tissue--altered synthesis and impaired hepatic secretion

Bile acid analysis of mild and severe cirrhotic liver showed that with the advancement of cirrhosis the concentration of chenodeoxycholic acid in liver tissue becomes higher, resulting in the lower ratio of cholic to chenodeoxycholic acid probably due to the progressive alteration of cholic and chenodeoxycholic acid synthesis with the advancement of liver cirrhosis. Bile acid analysis of paired liver and bile of severe cirrhosis showed that the ratio of cholic to chenodeoxycholic acid in liver was lower than that in bile or even with that in bile. This can be explained by postulating the impaired hepatic secretion of bile acids, especially chenodeoxycholic acid. The impaired secretion together with the relatively well preserved chenodeoxycholic acid synthesis results in the accumulation of chenodeoxycholic acid in liver tissue with cirrhosis.     

Transport of serum bile acids in patients with liver cirrhosis and hyperbilirubinaemia

From 12 patients with liver cirrhosis and hyperbilirubinaemia, the different conjugates of bilirubin and bile acids in the serum were separated and determined. The serum of the patients contained varying amounts of unconjugated bilirubin, which was not correlated to total serum bilirubin. No correlation between bilirubin conjugates and different conjugates of bile acids could be found, indicating different elimination processes for these substances. To examine whether a changed plasma transport of bile acids, which may contribute to the different excretion pattern of bilirubin and bile acids, occurs in liver cirrhosis, the bile acids in the different serum lipoprotein fractions were determined in seven of the patients. It was found that 40% of serum bile acids were bound to serum lipoproteins, despite decreased serum lipoprotein levels. The degree of lipoprotein binding of bile acids was not correlated to total serum bile acid concentrations. Cholic acid conjugates were present to a higher extent in the lipoprotein fractions than those of chenodeoxycholic acid or of deoxycholic acid. Determination was made of the distribution of individual conjugates between different lipoproteins and it was found that most of the glycine conjugates were present in high density lipoprotein, whereas the main part of sulphates and taurine conjugates were present in low density lipoprotein. These results indicate that a higher fraction of bile acids in liver cirrhosis is transported by lipoproteins in plasma, which may be of importance for the hepatic elimination of bile acids in cases with this disease.     

A radioimmunoassay of primary bile acid conjugates in human serum

Summary The fasting levels of chenodeoxycholic and cholic acid conjugates in various groups of patients [25 healthy subjects, 20 patients with chronic active liver disease (CALD), 21 patients with primary biliary cirrhosis (PBC) and 5 patients with cholesterol gallstones before and after chenodeoxycholic acid therapy] were determined by using a sensitive, accurate and precise radioimmunoassay. The results obtained with this method were compared with those of a gas-chromatographic method with methylation and acetylation of the bile acids using a 2 m × 0.4 cm glass column packed with 3% of AN 600 on Anakrom Q 110–120 mesh. The results obtained with both methods are in agreement; therefore the method used can be applied for quantitative analysis. In diagnosis, the measurement of serum bile acid levels, fasting or after meals, proved to be a very sensitive method, although further correlations are necessary with standard liver function tests.     

Influence of cholic and chenodeoxycholic acid on biliary cholesterol secretion in man

The interrelationships between biliary bile acid, lecithin and cholesterol secretion rates were studied druing depletion of the bile acid pool and during duodenal administration of cholic or chenodeoxycholic acid in thirteen patients 7-12 days after operation for uncomplicated gallstone disease. The mean lecithin secretion rate was signigicantly higher during cholic acid than during chenodeoxycholic acid infusion. The relationship between bile acid and cholesterol secretion rates was curvilinear, y = x/(a + bx) during bile acid pool depletion and during duodenal cholic acid infusion. At low bile acid secretion rates, during bile acid pool depletion and during cholic acid infusion, the lecithin secretion rate was significantly correlated to the cholesterol secreation rate. The bile acid and cholesterol secretion rates were not significantly correlated during chenodeoxycholic acid infusion. However, under this experimental condition a significant curvilinear relationship between lecithin and cholesterol secretion rates was found. The hepatic bile became unsaturated in cholesterol at significantly lower bile acid secretion rate during chenodeoxycholic acid infusion (10.7 +/- 0.3 mumol min-1) than during cholic acid infusion (15.6 +/- 0.5 mumol min-1).     

High molecular weight isoenzymes of alkaline phosphatase in human serum. Demonstration by cellulose acetate electrophoresis and physico-chemical characterization

A radioimmunoassay for the measurement of serum conjugated cholic acid has been developed. Antiserum to glycocholic acid coupled to bovine serum albumin was raised in rabbits. The antiserum was labelled with gly-co[³H] cholic acid and the bound label was separated from the free by precipitation using saturated ammonium sulphate. The amount of label bound decreased linearly with logarithmic increase in the amount of (non-active) glycocholic acid present (10–60 pmol). The cross reactivity of the common bile acids at 50% displacement of bound label was: taurocholic 100%; conjugated chenodeoxycholic acid 10%; conjugated deoxycholic 5%; cholic 10%; chenodeoxycholic and deoxycholic 2.5%. No cross-reactivity was detected with lithocholic acid or its conjugates, or with cholesterol. Between batch precision of the lowest standard concentration (0.1 μmol/l) was 14% and of the highest standard concentration (0.6 μmol/1) < 10%. It was not possible to obtain consistent data on unextracted sera and a simple one stage extraction procedure with ethanol was used. Fasting serum conjugated cholic acid concentrations in 8 laboratory personnel were 0.55–1.8 μmol/1. This method is a rapid and sensitive means for providing an indication of serum conjugated cholic acid concentrations but because of the cross reactivity properties of the antisera, it is not specific for conjugated cholic acid.     

Differential diagnostic value in hepatobiliary disease of serum conjugated bile acid concentrations and some routine liver tests assessed by discriminant analysis

Serum concentrations of glycine and taurine conjugates of cholic, chenodeoxycholic, and deoxycholic acid were measured with a HPLC-enzymatic assay in 104 patients with hepatobiliary diseases. The capability of discriminating between four major diagnostic categories by the serum bile acid concentrations, alone and in conjunction with five commonly used biochemical liver tests, was evaluated by stepwise linear discriminant analysis. The serum bile acid pattern alone was inferior to the routine liver tests in separating the diagnostic groups. When the two sets of analytes were combined, the generalized group distance (Rao's V) was significantly increased, showing that the serum bile acid pattern contained discriminatory information in addition to the routine liver tests. The gain in correct reclassification of patients obtained by adding the serum bile acids, however, was only marginal.     

Serum bile acids after a test meal in Crohn's disease

The serum levels of conjugated cholic and chenodeoxycholic acid have been studied before and during a 4 h period after the intake of a liquid test meal in seven control subjects and in fourteen patients with Crohn's disease. The concentrations of serum bile acids were determined by radioimmunoassay. The control group showed a postprandial increase of both conjugates with a return to the fasting level for cholic acid within 4 h. The chenodeoxycholic acid conjugate did not return to the fasting level within the test period. The serum bile acid concentration in Crohn's disease divided the patients in two groups; one group with decreased or normal fasting levels and low postprandial increase and another group with elevated fasting levels and a postprandial increase without return to the fasting levels within the test period.     

The effect of cholestyramine on the elimination of cholesterol as bile acids in patients with hyperlipoproteinaemia type II and IV

Abstract. The turnover of ¹⁴ C-cholic acid and ³ H-chenodeoxycholic acid was studied in hyperlipaemic patients before and during treatment with cholestyramine. — In five female patients with hyperlipoproteinaemia type IIa the pool size and turnover of cholic acid as well as the total formation of bile acids were significantly lower than in female normolipaemic controls. During treatment with cholestyramine, cholic acid synthesis rose 4–18 fold and the turnover of chenodeoxycholic acid increased about twice. In three of the patients these changes were associated with a normalization of the serum cholesterol levels. Mainly due to the high turnover of cholic acid, total bile acid synthesis was above the normal range in the five male patients with hyperlipoproteinaemia type IV. During treatment with cholestyramine, the serum lipids and cholic acid formation remained unchanged but the turnover of chenodeoxycholic acid increased by a factor of about two. This resulted in a decrease of the abnormally high initial ratio between the formation of cholic acid and chenodeoxycholic acid. The unequal response to cholestramine treatment is taken to be a further indication that hyperlipoproteinaemia type II and type IV are different entities of disease affecting cholesterol and bile acid metabolism.     

The Effect of Cholestyramine on the Elimination of Cholesterol as Bile Acids in Patients with Hyperlipoproteinaemia Type II and IV

Abstract. The turnover of ¹⁴C-oholic acid and ³H-chenodeoxycholic acid was studied in hyperlipaemic patients before and during treatment with cholestyramine. – In five female patients with hyperlipoproteinaemia type Ila the pool size and turnover of cholic acid as well as the total formation of bile acids were significantly lower than in female normolipae-mic controls. During treatment with cholestyramine, cholic acid synthesis rose 4–18 fold and the turnover of chenodeoxy-cholic acid increased about twice. In three of the patients these changes were associated with a normalization of the serum cholesterol levels. Mainly due to the high turnover of cholic acid, total bile acid synthesis was above the normal range in the five male patients with hyperlipoproteinaemia type IV. During treatment with cholestyramine, the serum lipids and cholic acid formation remained unchanged but the turnover of chenodeoxycholic acid increased by a factor of about two. This resulted in a decrease of the abnormally high initial ratio between the formation of cholic acid and chenodeoxycholic acid. The unequal response to cholest-ramine treatment is taken to be a further indication that hyperlipoproteinaemia type II and type IV are different entities of disease affecting cholesterol and bile acid metabolism.     

Bile acid synthesis and excretion following release of total extrahepatic cholestasis by percutaneous transhepatic drainage

Abstract. Urinary, biliary and serum bile acids were studied in three patients before and after percutaneous transhepatic drainage for total bile duct obstruction.
Before drainage high urinary excretion often different bile acids occurred. The percentage distribution was: cholic and chenodeoxycholic acid (66–86%), hyo-cholic (3–16%), 3β 12α-dihydroxy-5-cholenoic (3–6%) and 3β-hydroxy-5-cholenoic acid (2–8%). These acids were regularly found in serum. In addition small amounts (less than 2%) of norcholic, allocholic, 3β, 7α-dihydroxy-5β-cholanoic, 3α, 7α-dihydroxy-5α-cholanoic and lithocholic acid were excreted in urine. Trace amounts of these bile acids were found in serum.
After start of drainage biliary bile acid excretion increased rapidly during the first day, dropped to a minimum during the second or third day and then slowly increased again. In spite of normal volumes of bile produced, the total serum bile acids and the urinary excretion of bile acids remained increased during a drainage period of 19 days. The bile acids were of the same type as observed during cholestasis. In serum the increase was mainly due to high concentrations of chenodeoxycholic and 3β-hydroxy-5-cholenoic acid, as sulphate esters.
Glycine and taurine conjugates of cholic, chenodeoxycholic and hyocholic acid were mainly excreted in bile. Bile acid sulphate esters were only present in trace amounts in bile and were mainly excreted in urine. This, combined with low renal clearance, explains the elevated serum levels of sulphate esters of chenodeoxycholic and 3β-hydroxy-5-cholenoic acid conjugates.     

Equilibration of labelled and endogenous bile acids in patients with liver cirrhosis after administration of (24-14C)cholic and chenodeoxycholic acids

On separate occasions (24-14C)cholic acid and (24-14C)chenodeoxycholic acid were administered intravenously to patients with liver cirrhosis and the isotope excretion in urine and faeces monitored. Bile acids in serum, urine and faeces were extracted and separated into unconjugated bile acids, glycine- and taurine conjugates, glucuronides and sulphates. Individual bile acid conjugates were separated by high-performance liquid chromatography (HPLC) and the unconjugated bile acids were separated by gas-liquid chromatography (GLC) and identified by gas chromatography-mass spectrometry (GC-MS). Individual bile acid conjugates were quantified and their isotope contents determined. In serum, isotope contents declined rapidly during the first day, followed by a markedly slow rate of reduction. In accordance with this, the excretion of isotope from the patients was found to be very slow and the routes of bile acid excretion were changed, which resulted in an increased ratio of urine/faeces isotope excretion. Studies of the ratio of labelled to endogenous bile acid conjugates indicated that a continuous transformation of the labelled compounds occurred during the period of study. As judged from serum bile acids, conjugation to glycine- or taurine conjugates was rapid. The specific activities of labelled sulphate esters were consistently lower than for other conjugates during the 300-min observation period. During the first day, the urinary bile acids contained a high proportion of unconjugated labelled bile acids, which gradually disappeared. Labelled primary bile acids were slowly converted into microbial products, mainly 7-alpha dehydroxylated derivatives. The observed slow transformations resulted in a much delayed equilibration of labelled and endogenous bile acid derivatives, which invalidates isotope techniques for calculation of kinetic data of bile acid turnover. However, the observed very slow turnover of labelled bile acids in cirrhosis, owing to the persistent high rate of intestinal absorption and low capacity for urinary excretion, makes it possible for the intestinal flora to markedly change the composition of the bile acids in the pool. Studies of endogenous urinary and faecal bile acid excretion revealed the changed route of bile acid excretion with a high urinary/faeces ratio and the decreased synthesis of bile acids in cirrhosis.     

An Analysis of Lipoproteins, Bile Acids, and Red Cell Membranes Associated with Target Cells and Spur Cells in Patients with Liver Disease

Most patients with stable cirrhosis of the alcoholic have “target” red cells; however, a minority have “spur” cells and severe hemolytic anemia. These two syndromes were studied in 27 patients with target cells and 17 patients with spur cells, all of whom had advanced cirrhosis. The cholesterol and phospholipid content of red cell membranes effectively distinguished target cells from spur cells. Target cells alone were rich in lecithin, and both the cholesterol/phospholipid and cholesterol/lecithin mole ratios were greater in spur cells. The cholesterol/phospholipid mole ratio of both types of red cells correlated closely with the free cholesterol saturation of serum lipoproteins, as defined by the amount of free cholesterol relative to phospholipid and protein in these lipoproteins. Lecithin: cholesterol acyltransferase (LCAT) activity was decreased in most patients with target cells and spur cells; however, the relationship between this activity and the lipid abnormalities observed was weak. Serum bile acid levels also correlated poorly with serum and cell lipids. However, in patients with target cells the amount of cholic and deoxycholic acids in serum was approximately equal to the amount of chenodeoxycholic acid, whereas in patients with spur cells chenodeoxycholic acid (the precursor of lithocholic acid) predominated.     

Bile acids in serum, ultrafiltrate of serum and saliva from patients with cholestatic jaundice

Individual bile acid conjugates in serum, the ultrafiltrate of serum and in saliva from patients with cholestatic jaundice were determined by gas-liquid chromatography. The bile acid concentration in the ultrafiltrate was 4-20% of that in serum and had also a different composition, with higher ratio of cholic to chenodeoxycholic acid. In saliva, the bile acid concentration was 1-8% of that in serum and the composition was different due to a higher proportion of unconjugated cholic acid. The results indicate that neither the bile acid concentration in the ultrafiltrate, nor that in saliva corresponds to the non-protein-bound bile acids in serum.     

Determination of cholic acid and chenodeoxycholic acid pool sizes and fractional turnover rates by means of stable isotope dilution technique, making use of deuterated cholic acid and chenodeoxycholic acid

A procedure is described for the simultaneous determination of cholic acid and chenodeoxycholic acid pool sizes and fractional turnover rates. After oral administration of known amounts of 11,12-dideuterated chenodeoxycholic acid and 2,2,4,4-tetradeuterated cholic acid, the ratios of chenodeoxycholic acid-D2/chenodeoxycholic acid and cholic acid-D4/cholic acid are measured in consecutive serum samples, after which fractional turnover rates and pool sizes of chenodeoxycholic acid and cholic acid are determined arithmetically. In 7 healthy volunteers pool sizes for chenodeoxycholic acid and cholic acid were 22.9 +/- 7.8 and 24.1 +/- 11.7 mumol/kg, respectively. The corresponding values for the fractional turnover rates were 0.23 +/- 0.10 and 0.29 +/- 0.12/day. After oral administration of the labelled bile acids in capsule, the obtained pool sizes were significantly higher than after administration in a bicarbonate solution. Bile acid kinetics were also performed in a patient suffering from a cholesterol synthesis deficiency and in a patient very likely suffering from a bile acid synthesis deficiency. Furthermore, the kinetics of the intestinal absorption and hepatic clearance of unconjugated bile acids have been investigated in 2 healthy subjects.     

Bile-acid metabolism and the liver.

Primary bile acids are exclusively synthesize, and all bile acids are conjugated, in the liver. When hepatic function is altered by disease, not surprisingly the bile-acid metabolism reflects this change. 1. In chronic liver disorders the glycine: taurine ratio of serum and bile is reduced. 2. In cases of relapsing acute hepatitis and relapsing chronic active hepatitis the serum bile acids are increased, providing an aid to early diagnosis. Their pattern often distinguishes chronic cholestatic conditions from Laennec's cirrhosis. 3. In chronic liver disease, the concentration of intraduodenal bile acids is reduced; when the reduction is severe, this probably accounts for co-existent steatorrhoea. 4. In Laennec's cirrhosis, both the synthesis and pool size of cholic acid are markedly depressed; in primary biliary cirrhosis, however, preliminary data indicate a decrease in the chenodeoxycholic acid component.     

In vivo and vitro studies on formation of bile acids in patients with Zellweger syndrome. Evidence that peroxisomes are of importance in the normal biosynthesis of both cholic and chenodeoxycholic acid.

The last step in bile acid formation involves conversion of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) into cholic acid and 3 alpha,7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA) into chenodeoxycholic acid. The peroxisomal fraction of rat and human liver has the highest capacity to catalyze these reactions. Infants with Zellweger syndrome lack liver peroxisomes, and accumulate 5 beta-cholestanoic acids in bile and serum. We recently showed that such an infant had reduced capacity to convert a cholic acid precursor, 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol into cholic acid. 7 alpha-Hydroxy-4-cholesten-3-one is a common precursor for both cholic acid and chenodeoxycholic acid. Intravenous administration of [3H]7 alpha-hydroxy-4-cholesten-3-one to an infant with Zellweger syndrome led to a rapid incorporation of 3H into biliary THCA but only 10% of 3H was incorporated into cholic acid after 48 h. The incorporation of 3H into DHCA was only 25% of that into THCA and the incorporation into chenodeoxycholic acid approximately 50% of that in cholic acid. The conversion of intravenously administered [3H]THCA into cholic acid in another infant with Zellweger syndrome was only 7%. There was a slow conversion of THCA into 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-C29-dicarboxylic acid. The pool size of both cholic- and chenodeoxycholic acid was markedly reduced. Preparations of liver from two patients with Zellweger syndrome had no capacity to catalyze conversion of THCA into cholic acid. There was, however, a small conversion of DHCA into chenodeoxycholic acid and into THCA. It is concluded that liver peroxisomes are important both for the conversion of THCA into cholic acid and DHCA into chenodeoxycholic acid.     

Postprandial variations in serum bile acid levels in humans free of hepatic or intestinal diseases (author's transl)

The levels of unsulfated, free or conjugated cholic, deoxycholic and chenodeoxycholic acids were measured using gas chromatography in 39 humans free of hepatic or intestinal diseases before and 10, 60, 120 and 180 min after ingestion of a standard meal. The probable maximal levels were determined with an error risk lower than 0.05. In fasting subjects, the observed values are comparable with those obtained by other authors working with gas chromatography or radioimmunoassay. Meal ingestion does not influence in the same way the serum levels of the various bile acids: the chemodeoxycholic serum level rose significantly in all cases whereas cholic and deoxycholic serum levels rose only in two-thirds of observed subjects; 60 and 120 min after the meal for chenodeoxycholic acid, and only 60 min after the meal for cholic acid, the mean values are significantly higher than the fasting ones; 120 min after the meal, the chenodeoxycholic and total bile acid probable maximal levels (respectively 7.4 and 10.3 micrometer) are twice the fasting ones. The cholic to chenodeoxycholic serum level ratio is nearly always lower than 1 but may reach 3. On the basis of these results, the validity and efficacy of the exploration tests based on serum bile acid level determinations are discussed.     

Bile acid transport by serum lipoproteins in patients with extrahepatic cholestasis

Individual bile acid conjugates were determined by gas-liquid chromatography in very low density (VLDL), low density (LDL) and high density (HDL) lipoprotein fractions obtained by sequential ultracentrifugation of serum from cholestatic patients. In the lipoproteins were found 16-48% of the serum bile acids. In LDL, the amount found was twice that in VLDL and HDL together. More cholic than chenodeoxycholic acid was detected in the lipoproteins. The occurrence of bile acids in lipoproteins might be of importance for the transport of bile acids from blood to peripheral tissues during cholestasis.     

Serum bile acid profile in thyroid dysfunction and effect of medical treatment

1. Serum non-esterified bile acid profile was examined in patients with thyroid dysfunction. Sixteen hyperthyroid patients, six hypothyroid patients, nine patients taking thyroid or antithyroid drugs and 26 healthy controls were studied. The medicated patients were euthyroid when serum samples were collected. Bile acid concentration was determined by the simplified microassay method involving mass fragmentation spectrometry. 2. The sum of the concentrations of the individual bile acids was not significantly different among the four groups. However, the composition of bile acid reflected the thyroid function. The most prominent bile acid was deoxycholic acid in the hypothyroid patients and chenodeoxycholic acid in the hyperthyroid patients. The serum bile acid profile of medically treated patients was similar to that of normal controls. The ratio of the sum of deoxycholic and cholic acid to that of lithocholic and chenodeoxycholic acid was found to be a good indicator of thyroid function, while the ratio of cholic acid to chenodeoxycholic acid correlated poorly with it. 3. The characteristic effect of thyroid hormone on the serum bile acid composition in man was the shift from the 'family' of cholic acid to that of chenodeoxycholic acid. This is in agreement with experimental results in the rat, and suggests a specific action of thyroid hormone on the hydroxylating enzymes involved in the conversion of cholesterol into bile acids.     

改良的肝硬化门脉高压伴腹水大鼠模型

目的 :建立一种制备方法简便、具有高存活率及成模率的肝硬化、门脉高压伴腹水的大鼠动物模型。方法 :80只重量为 2 0 0～ 2 5 0 g的雄性SD大鼠 ,随机选取 10只为正常对照组 ,其余 70只为肝硬化腹水造模组。造模组大鼠的处理 ,苯巴比妥溶液 (35mg % )作为饮用水诱导 1周。第 2周始每周给予 4 0 %CCl4-橄榄油溶液皮下注射 2次 ,每次剂量为 0 .2ml / 10 0g ,首剂加倍 ,第 9周始改为每 2周 3次直至第 14周。结果 :第 14周 ,肝硬化腹水造模组 70只大鼠存活 37只 ,其中 36只存在腹水。肝硬化腹水造模组的大鼠肝脾明显肿大 ;肝表面凹凸不平 ,可见大小结节 ;组织学检查显示肝硬化、假小叶的形成 ;门静脉压力较正常对照组明显升高 (17.6 7± 3.4 7vs 8.6 7± 1.0 3cmH2 O ,P <0 .0 1) ;血清总胆红素、谷丙转氨酶及谷草转氨酶较正常对照组明显升高 (P <0 .0 5 ) ,而血清白蛋白较之正常对照组明显降低 (P <0 .0 1)。结论 :本实验经对CCl4加苯巴比妥联合造模法加以改良 ,获得了一种肝硬化门脉高压伴腹水的大鼠模型 ,为进一步研究创造条件