Bile acid profiles in urine of patients with liver diseases

Abstract. Quantitative gas chromatography-mass spectrometry was used to study the metabolic profiles of unconjugated, conjugated and sulphated bile acids in urine of patients with intermittent intrahepatic cholestasis of unknown aetiology, cirrhosis of the liver, primary biliary cirrhosis, viral and toxic hepatitis and extrahepatic cholestasis. A large number of bile acids was present which can broadly be classified into four groups: cholic and chenodeoxycholic acids constituted between 49·4% and 77·9% of the total bile acids (mean values of the groups); deoxycholic and other 3,12-disubstituted bile acids between 1·3% and 12·3%, monohydroxy bile acids between 6·7% and 14·4% and bile acids hydroxylated at C-1 or C-6 between 4·6% and 14·6%. The high proportion of bile acids from the latter group, and the presence of tetrahydroxylated bile acids, clearly distinguished hepatic disease from the normal state. The metabolic profiles were very variable and there were few consistent differences between the groups of diseases studied. Norcholic acid constituted a significantly higher percentage of the total bile acids in cirrhotic patients (6·2 ± 6·8%) than in non-cirrhotic patients (1·3±1·8%, P<0·001). With this exception, no profile was specific for any type of intra- or extra-hepatic cholestasis. The excretion rates of the major l-hydroxylated bile acids were positively correlated to each other. The same was true for the major 6-hydroxylated bile acids. This may indicate that cholic, chenodeoxycholic and deoxycholic acids act as substrates for common 1- and 6-hydroxylating enzymes. Possibly the taurine conjugates are preferred substrates since 1-hydroxylated bile acids and hyocholic acid were found mainly in this fraction. A positive correlation between the excretion of sulphated 3β-hydroxy-5-cholenoic acid and 3β,12α-dihydroxy-5-cholenoic acid indicates a direct metabolic relationship between these compounds. Confirming previous data, a high proportion of bile acids was sulphated. The degree of sulphation increased with decreasing number of hydroxyl groups, reaching 100% for the monohydroxy and most of the dihydroxy acids. Tetrahydroxycholanoates were not sulphated, and sulphation of trihydroxycholanoates was positively correlated to the renal bile acid excretion rate. Bile from patients with intermittent intrahepatic cholestasis did not contain the tetrahydroxylated bile acids present in urine. Hyocholic acid was a very minor, mainly taurine conjugated, bile acid. Monohydroxy bile acids were usually below the detection limit. These data do not support the hypothesis that lithocholic acid participates in the initiation or perpetuation of intermittent intrahepatic cholestasis of unknown aetiology.     

Bile Salt Glucuronides: Identification and Quantitative Analysis in the Urine of Patients with Cholestasis

Glucuronides of lithocholate, chenodeoxycholate and cholate were synthesized enzymatically and characterized by thin layer chromatography, column chromatography and specific enzymatic hydrolysis. Bile salt glucuronides were quantitatively analysed in thr urine of patients with intra- and extra- hepatic cholestasis and were found to be present in 19 out of 20 patients studied. Our patients with intrahepatic cholestasis excreted 8.9 mg non-sulphated and non-glucoronidated bile salts, 18.2 mg sulphated bile salts and 7.2 mg glucuronidated bile salts. The patients with extrahepatic cholestasis excreted 14.7 mg non-sulphated and non-glucuronidated bile salts, 20.7 mg sulphated bile salts and 4.7 mg glucuronidated bile salts. These findings indicate that glucuronidation of bile salts occurs in man and represents a metabolic pathway in patients with cholestasis.     

Conjugation, metabolism and excretion of [24-14C] chenodeoxycholic acid in patients with extrahepatic cholestasis before and after biliary drainage-analysis of conjugated bile acids by HPLC

[24-14C] chenodeoxycholic acid (CDC) was given to patients with total extrahepatic cholestasis two or three days before an external drainage was made, and excretion of the isotope in urine and bile followed. Bile acids were group-separated by anion exchange chromatography on DEAP-Sephadex LH-20 and the individual conjugates isolated by HPLC. 51.0-75.4% of the administered isotope was excreted; 16.2-29.9% as sulphates, 0.1-2.4% as glucuronides and 20.7-58.7% as glycine and taurine conjugates. 5.2-21.0% of excreted isotope consisted of transformation products of CDC, mainly cholic acid, hyocholic acid and ursodeoxycholic acid. Labelled urinary sulphates were the 3-sulphates of glycochenodeoxycholic and taurochenodeoxycholic acid. During cholestasis the renal clearance was about ten times higher for the sulphates compared with the non-sulphated conjugate. The clearance of glycine conjugates and their sulphates was of the same magnitude as that of the corresponding taurine conjugates. During the biliary drainage period, most of the labelled sulphates were excreted in urine, while most of the glycine and taurine conjugates were excreted in bile.     

Evidence for renal control of urinary excretion of bile acids and bile acid sulphates in the cholestatic syndrome

1. The bile acids and bile acid sulphates in the urine, serum and bile of eight cholestatic patients were studied quantitatively by gasliquid chromatography and gas-liquid chromoatography/mass spectrometry. 2. The primary bile acids (cholic acid and chenodeoxycholic acid) comprised on average 94% of the total bile acids in bile, 70% in the serum and 64% in urine. 3. The percentage composition of bile acids in bile was relatively constant and was not influenced by the degree of cholestasis. In contrast, in the serum only the primary bile acids were increased, the concentrations of the secondary bile acids (deoxycholic acid and lithocholic acid) and the minor bile acids remaining constant. 4. The data do not support the hypothesis that monohydroxy bile acids accumulate in cholestasis and are related to the pathogenesis of this syndrome. 5. The pattern of bile acid urinary excretion was similar to that in the serum. But in one patient, 3alpha, 7beta, 12alpha-trihydroxy-5beta-cholan-24-oic acid was a principal urinary bile acid, although very low concentrations of the compound were found in that patient's serum, suggesting that some of the minor bile acids in urine may originate by epimerization in the kidney. 6. In bile only a small proportion of the bile acids was sulphated (range 2.1-4.6%) and in serum the degree of sulphation was very variable (9-50%). However, in urine, sulphate esters accounted for a large proportion of the total bile acids (33-72%). 7. The output of bile acid sulphate in the urine was related to the urine total bile acid output but the serum concentration of bile acid sulphate remained relatively constant. Consequently, in contrast to the non-sulphated bile acids, whose renal clearance was relatively constant, the renal clearance of sulphated bile acids was directly related to the urine total bile acid output. This finding is inconsistent with the earlier hypothesis that their predominance in urine was due to a high renal clearance. It may indicate renal synthesis of some of the bile acid sulphates in the urine and/or inhibition of active renal tubular reabsorption of sulphated bile acids by non-sulphated bile acids.     

Rapid diagnosis of Zellweger syndrome and infantile Refsum's disease by fast atom bombardment--mass spectrometry of urine bile salts

A method is described for the rapid determination of urinary bile salt profiles by fast atom bombardment--mass spectrometry (FAB-MS). Urine was passed through a reverse-phase octadecylsilane bonded silica cartridge and the bile salts eluted with methanol. Negative ion FAB spectra could be obtained from the equivalent of 10 microliter of urine loaded onto the target probe with glycerol as matrix. In samples from normal infants and children bile salt peaks were rarely detectable above the background whereas peaks produced by steroid sulphates and glucuronides and bile alcohol glucuronides could usually be identified. In samples from infants and children with cholestasis the major peaks were produced by the taurine and glycine conjugates of di-, tri- and tetrahydroxycholanoic acids (and their monosulphates). In samples from patients with Zellweger syndrome and infantile Refsum's disease, a unique ion at m/z 572 indicated the presence of taurine-conjugated tetrahydroxy-cholestanoic acid(s). The amide linkage to taurine was cleaved by alkaline hydrolysis but not by cholylglycine hydrolase. Capillary gas chromatography--mass spectrometry (GC-MS) of the bile acids liberated by alkaline hydrolysis indicated the presence of at least two nuclear-tetrahydroxylated cholestanoic acids, probably the 6 alpha- and 1 beta-hydroxylated derivatives of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid.     

Biotransformation of orally administered ursodeoxycholic acid in man as observed in gallbladder bile, serum and urine

Abstract. The aim of this study was to evaluate the biotransformation of orally administered ursodeoxycholic acid in man. The distribution of ursodeoxycholic acid and its metabolites in gallbladder bile, in serum and in urine with emphasis on separation of their unconjugated, amidated and sulfated species in particular, was investigated. Seven gallstone patients were given 750 mg of ursodeoxycholic acid daily for 2–3 weeks. Six gallstone patients who did not receive ursodeoxycholic acid served as controls. Ursodeoxycholic acid became the major bile acid in gallbladder bile contributing 43% to total bile acids. 2% of biliary ursodeoxycholic acids were in the unconjugated form, 87% in the amidated form and 11% in the sulfated form. Iso-ursodeoxycholic acid was found in bile in small amounts and was present only as the sulfated species and not as the amidated one. Other metabolites of ursodeoxycholic acid tentatively identified in bile were 1β, 12β, 6α- and 21,22-hydroxylated derivatives of ursodeoxycholic acid. Lithocholic acid in bile tended to increase under ursodeoxycholic acid treatment and was positively correlated to ursodeoxycholic acid. The concentration of cholic acid in bile decreased significantly whereas the levels of deoxycholic acid and chenodeoxycholic acid did not change. Total bile acid concentration in serum and excretion of bile acids in urine increased from 5.4 ± 1.1 to 18.4 ± 9.5 μmol l^-1 (mean ± SD, P < 0.005) and from 5.6 ± 1.3 to 13.1 ± 7.9 μmol g^-1creatinine (mean ± SD, P < 0.05) after ursodeoxycholic acid ingestion mainly due to spillover and excretion of ursodeoxycholic acid. Ursodeoxycholic acid became the major bile acid in serum and urine contributing 46% and 50% to total bile acids. 14% ursodeoxycholic acid in serum were in the unconjugated form, 42% in the amidated form and 45% in the sulfated form; the percentages in urine were 11%, 23% and 66%. Iso-ursodeoxycholic acid was higher in serum and urine than in bile and contributed 16% and 8% to total bile acids. Iso-ursodeoxycholic acid was present in serum and urine only as the unconjugated and sulfated species. Other iso-bile acids and 3β-hydroxy-5-cholenoic acid were found in bile only in traces, but contributed 8% to total bile acids in serum and 10% in urine. In serum and urine the sulfated form of lithocholic acid prevailed and was significantly enhanced after ursodeoxycholic acid ingestion. Further metabolites of ursodeoxycholic acid in urine were tentatively identified to be hydroxylated at postitions 1β, 5α, 6α and 22 and contributed about 10–15% of urinary UDCA.     

The measurement of sulphated and non-sulphated bile acids in serum using gas-liquid chromatography.

A method is described to assay sulphated and non-sulphated bile acids in serum using gas-liquid chromatography. Previously described techniques have been substantially modified to allow analysis of free and conjugated salts of the four major bile acids with particular care to ensure quantitative recoveries of lithocholic acid, its conjugates and sulphate esters. Losses of lithocholic acid inherent in some methods have been reduced by avoidance of column chromatography with alumina and extraction of lipid contaminants into heptane. Assay of the proportion of serum bile acids present as sulphate esters is achieved by the routine use of column chromatography to separate sulphated bile acids from non-sulphated bile acids followed by solvolysis of the sulphated bile acids before deconjugation. Careful selection of the conditions of strong alkaline hydrolysis ensures deconjugation of all bile salt conjugates including lithocholic conjugates which are not completely hydrolysed in weaker alkaline solutions. The trifluoroacetate derivatives of the methyl esters of the bile acids are chromatographed using 5-beta-cholanic acid as an internal standard with clear separation of the four major bile acids from the internal standard. In 10 fasting control subjects the mean serum total bile acid concentration was 5.3 muM (RANGE 1.1-16.4) including 0.7 mum sulphated bile acid (range 0-1.8). In 10 patients with acute viral hepatitis the total bile acid concentration was elevated in some but normal in others (mean 44.9 muM, range 2.7-80.3). The percentage of the total bile acid sulphated was not significantly different in the hepatitis patients compared to controls (controls 13%, range 0-35; hepatitis 23%, range 0-52). Lithocholic acid made up 13% of the total bile acid in controls (0-32%) and 18% in hepatitis patients (0-53%). Most of this lithocholic acid was sulphated (controls 81%, range 30-100; hepatitis 67%, range 37-100). Unconjugated bile acids were demonstrated in the serum of a few patients with acute viral hepatitis but in no control subjects.     

The nature of urinary bile acid conjugates in patients with extrahepatic cholestasis

Urinary bile acids from patients with extrahepatic cholestasis were extracted with Sep-pak C18 cartridges and group separated on diethylaminohydroxypropyl Sephadex LH-20. The nature of the different conjugates of cholic and chenodeoxycholic acid in the fractions was studied after further separation by preparative thin-layer chromatography. The free and glycine-conjugated bile acids were quantified by capillary gas chromatography and identified by gas chromatography-mass spectrometry (GC/MS). Taurine conjugates were split with cholylglycine hydrolase and the liberated free bile acids analysed by GC/MS. Sulphate esters were hydrolysed with Helix pomatia and the resulting bile acid derivatives were analysed as above. After hydrolysis with cholylglycine hydrolase, the glucuronides of the unconjugated bile acids were separated and identified by GC/MS. Amino acid analysis of the different fractions revealed that glycine and taurine were the only amino acids present in connection with cholic and chenodeoxycholic acid. Large amounts of monosulphated bile acid conjugates were present but no disulphates. Only 3-sulphates were found. Both sulphates and glucuronides were found exclusively as glycine or taurine conjugates and no such derivatives of unconjugated bile acids were isolated. The isolated conjugates were split either by a combination of acid solvolysis and alkaline hydrolysis or by Helix pomatia and cholylglycine hydrolase.     

Urinary excretion of bile alcohols in normal children and patients with α1-antitrypsin deficiency during development of liver disease

Abstract. Healthy infants and children were found to excrete bile alcohol glucuronides in urine. Following isolation and hydrolysis, the bile alcohols were estimated by capillary gas-liquid chromatography. The daily urinary excretion of the major compound, 27-nor-5 β -cholestane-3α,7α,12α,24ξ,25ξ-pentol (a C₂₆ bile alcohol), ranged from 0·1 to 1·1 μmol/24 h per m² body surface area for healthy infants and children. Two groups of patients with α₁-antitrypsin deficiency (phenotype PiZ) were also studied during infancy and childhood, and biochemical liver function tests and liver morphology were compared to the excretion of bile alcohols. The highest excretion of the C₂₆ bile alcohol in urine was found in patients with α₁-antitrypsin deficiency and juvenile cirrhosis (2·1–8·4 μmol 24 h^-1 m^-2) regardless of preceding neonatal cholestasis. Patients with α₁-antitrypsin deficiency, neonatal cholestasis and subsequent fibrosis or normal liver morphology excreted bile alcohols within the normal range. The C₂₆ bile alochol constituted an average of 36% of the total bile alcohols in forty-three urine samples. This percentage was about the same in the three groups studied. The findings suggest that determination of urinary bile alcohols may be a valuable non-invasive diagnostic tool for patients with or at risk of developing liver cirrhosis.     

Protective effects of 6-ethyl chenodeoxycholic acid, a farnesoid X receptor ligand, in estrogen-induced cholestasis

The farnesoid X receptor (FXR), an endogenous sensor for bile acids, regulates a program of genes involved in bile acid biosynthesis, conjugation, and transport. Cholestatic liver diseases are a group of immunologically and genetically mediated disorders in which accumulation of endogenous bile acids plays a role in the disease progression and symptoms. Here, we describe the effect of 6-ethyl chenodeoxycholic acid (6-ECDCA or INT-747), a semisynthetic bile acid derivative and potent FXR ligand, in a model of cholestasis induced by 5-day administration of 17alpha-ethynylestradiol (E(2)17alpha) to rats. The exposure of rat hepatocytes to 1 microM 6-ECDCA caused a 3- to 5-fold induction of small heterodimer partner (Shp) and bile salt export pump (bsep) mRNA and 70 to 80% reduction of cholesterol 7alpha-hydroxylase (cyp7a1), oxysterol 12beta-hydroxylase (cyp8b1), and Na(+)/taurocholate cotransporting peptide (ntcp). In vivo administration of 6-ECDCA protects against cholestasis induced by E(2)17alpha. Thus, 6-ECDCA reverted bile flow impairment induced by E(2)17alpha, reduced secretion of cholic acid and deoxycholic acid, but increased muricholic acid and chenodeoxycholic acid secretion. In vivo administration of 6-ECDCA increased liver expression of Shp, bsep, multidrug resistance-associated protein-2, and multidrug resistance protein-2, whereas it reduced cyp7a1 and cyp8b1 and ntcp mRNA. These changes were reproduced by GW4064, a synthetic FXR ligand. In conclusion, by demonstrating that 6-ECDCA protects against E(2)17alpha cholestasis, our data support the notion that development of potent FXR ligands might represent a new approach for the treatment of cholestatic disorders.     

Isolation and quantification of nonsulfated and sulfated bile acids in the feces

A method is described for the qualitative and quantitative analysis of non-sulphated and sulphated bile acids in faeces. After extraction and preliminary purification, the faecal bile acids are separated by liquid-gel-chromatography (DEAP-Sephadex LH20) into free, conjugated and sulphated bile acids; these are quantitated separately (after solvolysis and hydrolysis), and the individual bile acids are analysed by gas-liquid-chromatography. The validation both of the individual analysis steps and the overall procedure by adding bile acid standards to the faecal homogenates showed a good reproducibility and a reliable separation of non-sulphated and sulphated bile acids. Using the described method, the excretion of total faecal bile acids in 15 control subjects was 3.85 mg/g dry stool, consisting of 10.4% primary bile acids and 89.6% secondary bile acids. 92.9% of faecal bile acids were in the free form, only 2.7% in the conjugated form, and 4.4% as sulphated bile acids.     

Urinary excretion of bile acids during acute administration in man

Six healthy subjects, 45-72 years old, received a 10-day feeding of 750 mg of two of the following bile acids: deoxycholate (DCA), chenodeoxycholate (CDCA), cholate (CA), hyodeoxycholate (HDCA), ursodeoxycholate (UDCA), and ursocholate (UCA). The urinary excretion of total bile acids was low during administration of lipophilic bile acids (DCA and CDCA), when serum levels show low postabsorption peaks. Instead, hydrophilic bile acids (UDCA and above all HDCA) were heavily excreted in the urine as sulphates and glucuronides, and serum levels reach high values. Only UCA, strongly hydrophilic, was predominantly excreted as unconjugated fractions. Thus, the physicochemical properties of bile acids (as measured by both the partition between octanol and water, and the water solubility) were factors that influenced the route of bile acid elimination from the body, whereas their conjugation was not always requested for urinary excretion.     

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.     

Bile acid N-acetylglucosaminidation. In vivo and in vitro evidence for a selective conjugation reaction of 7 beta-hydroxylated bile acids in humans.

The aim of this study was to define whether N-acetylglucosaminidation is a selective conjugation pathway of structurally related bile acids in humans. The following bile acids released enzymatically from N-acetylglucosaminides were identified: 3 alpha,7 beta-dihydroxy-5 beta-cholanoic (ursodeoxycholic), 3 beta, 7 beta-dihydroxy-5 beta-cholanoic (isoursodeoxycholic), 3 beta,7 beta-dihydroxy-5 alpha-cholanoic (alloisoursodeoxycholic), 3 beta,7 beta-dihydroxy-5-cholenoic, 3 alpha,7 beta,12 alpha-trihydroxy-5 beta-cholanoic, and 3 alpha,6 alpha,7 beta-trihydroxy-5 beta-cholanoic acids. The selectivity of conjugation was studied by administration of 0.5 g ursodeoxycholic (UDCA) or hyodeoxycholic (HDCA) acids, labeled with 13C, to patients with extrahepatic cholestasis, and of 0.5 g of 13C-labeled chenodeoxycholic acid (CDCA) to patients with extra- or intrahepatic cholestasis. After administration of [24-13C]-CDCA, labeled glucosides, and the glucuronide of CDCA were excreted in similar amounts. Labeled N-acetylglucosaminides of UDCA and isoUDCA were also formed. When [24-13C]-UDCA was given, 13C-label was detected in the N-acetylglucosaminide, the glucosides, and the glucuronide of UDCA, and in the N-acetylglucosaminide of isoUDCA. In the patient studied, 32% of the total UDCA excreted in urine was conjugated with N-acetylglucosamine. In contrast, 96% of the excreted amount of [24-13C]HDCA was glucuronidated, and 13C-labeled glucosides but no N-acetylglucosaminide were detected. The selectivity of N-acetylglucosaminidation towards bile acids containing a 7 beta-hydroxyl group was confirmed in vitro using human liver and kidney microsomes and uridine diphosphate glucose (UDP)-N-acetylglucosamine. These studies show that N-acetylglucosaminidation is a selective conjugation pathway for 7 beta-hydroxylated bile acids.     

Influence of Portacaval Shunt on Bile Formation in the Rat*

Abstract. Bile flow and bile acid secretion were measured in rats 21 to 28 days after a portacaval shunt and in sham-operated and normal animals. The following results were obtained. (1) Bile flow was significantly lower (6.65±SEM 0.36 μl min^-1-100 g^-l) in the shunted rats than in the shamoperated animals (8.21 ± SEM 0.21 μl min^-1 100 g^-1; P < 0.01). (2) Bile acid excretion was not significantly different in the shunted rats (0.27±SEM 0.03μmol-min^-1100g^-1) and the sham-operated rats (0.26±SEM 0.02 μmol-min^-1 -100 g^-1; NS). (3) During bile acid infusions, there was a linear relationship between bile flow and bile acid excretion in both groups of animals. The slope of the relationship was similar, suggesting that the osmotic activity of the bile acids was not modified in the shunted animals, and the bile acid-independent flow, estimated by the extrapolation of this relationship to a zero bile acid excretion, was significantly lower in the rats with a portacaval shunt (5.20±SEM 0.40 μ min^-1 100 g^-1) than in the sham -operated animals (6.50±SEM 0.30 μl min^-1 100 g^-1; P < 0.02). (4) The liver weight was significantly lower in the rats with a portacaval shunt than in the sham-operated animals and there was a parallel decrease of liver weight (–17%) and of the bile acid-independent flow (–22%). No difference was found between the sham-operated rats and the normal rats. It is concluded that portacaval shunt in the rat results in a decreased bile flow, due to a decrease in the bile acid-independent flow. Since bile acid secretion rate remained unchanged, it is suggested that the secretion of bile acids on the one hand and the bile acid-independent flow on the other are regulated by separate mechanisms.     

Urinary bile acids in late pregnancy and in recurrent cholestasis of pregnancy

The metabolic profiles of urinary bile acids in pregnant women in the last trimester and patients with recurrent intrahepatic cholestasis of pregnancy (RCP) were studied. Following separation according to mode of conjugation, about thirty different bile acids were quantitatively analysed by gas chromatography-mass spectrometry. In all patients the sulphate fraction comprised 50--90% of the total bile acids. In RCP a shift from glycine to taurine conjugation was noted to together with a slight relative increase in sulphation. A ten- to hundred-fold increase in cholic and chenodeoxycholic acids was seen in RCP, the increase being mainly in the sulphate fraction. Tetrahydroxylated bile acids, tentatively regarded as 1- and 6-hydroxylated products of cholic acid, were quantitatively important in patients with RCP. The relative amounts of the secondary bile acids, deoxycholic and lithocholic acids, decreased with increasing cholestasis. Metabolites hydroxylated at C-6 were common, and the excretion of hydroxylated at C-6 were common, and the excretion of hyocholic acid was positively correlated to that of chenodeoxycholic acid. An increase in the excretion of 5 alpha-configurated bile acids in RCP was noted. A positive correlation between the excretion of 3 beta-hydroxy-5-cholenoic acid and 3 beta,12 alpha-dihydroxy-5-cholenoic acid indicates a metabolic relationship between the two compounds. Because of the relatively small amounts of lithocholic and 3 beta-hydroxy-5-cholenoic acids in patients with RCP, these compounds do not seem to be of pathogenetic importance in this type of cholestasis.     

Pharmacokinetics of fusidic acid after a single dose of a new paediatric suspension

The pharmacokinetics of fusidic acid (Fucidine®, Leo Laboratories) were studied in 10 children after single oral dosing with 20 mg/kg of a new bananaflavoured paediatric suspension (titrating at 50 mg/ ml). Nine blood samples were drawn from each child at 0, 1, 2, 3, 6, 8, 12, 24 and 48 h following dosing with the antibiotic. Serum fusidic acid levels were measured by high–performance liquid chromatography (HPLC). A model–independent method was used for the pharmacokinetic analysis. Results were compared with those obtained after dosing eight healthy adult volunteers with 500 mg of sodium fusidate by parenteral administration (infusion) then per os. The acceptability of the single dose was good. The terminal elimination half–life t_1/2 (h) and the mean residence time (MRT, h) of fusidate were similar to those determined in healthy adults after oral dosing, i.e. 16–0 ± 14–5 versus 16–0 ± 3–5 and 17–7 ± 12–1 versus 17–7 ± 2–5, respectively. In contrast, the oral bioavailability of the suspension (F_approx, %) was relatively low: of the order of 225 versus 91–0% for tablets in the healthy adult, which justifies the use of a relatively higher dose in the child. This led to the calculation of an estimated total clearance (CI_est, ml/min) significantly less than that in the healthy adults, while the estimated apparent volume of distribution (V_d, litre/kg) was significantly increased (104 ± 91 versus 218 ± 21 and 073 ± 053 versus 030 ± 004, respectively). Fusidic acid is normally excreted in metabolized form (98%). The decrease in clearance could be attributed to the almost immediate saturation of liver enzymes in immature infants. An increased initial volume of distribution could result from less protein binding of fusidic acid. Graphic simulations show that the administration of a dose of 20 mg/kg every 12 h results in effective serum levels being obtained from the outset. A treatment schedule involving three doses per day (08.00, 12.00, 20.00 hours) is also theoretically satisfactory.     

Cholestasis induced by sulphated glycolithocholic acid in the rat: protection by endogenous bile acids

Sulphated glycolithocholic acid (SGLC) causes cholestasis in experimental animals, despite its sulphated form. In the present study, the cholestatic potency and the pharmacokinetics of SGLC were investigated in rats under two conditions: (a) in the presence of an intact circulating bile acid pool and (b) after exhaustion of the bile acid pool by 24 h of bile diversion. Intravenous administration of SGLC (8 mumol/100 g body weight) to rats with an intact bile acid pool did not cause cholestasis. However, biliary phospholipid and cholesterol concentrations were reduced by 40% and 29% respectively during the first hour after administration. When the same dose of the bile acid was injected in rats with a 24 h biliary drainage, a complete cessation of bile production was observed within 1 h. Twelve hours after the onset of cholestasis, bile production gradually increased again, showed a marked overshoot, and reached control levels after 3 days. In the recovery phase, biliary phospholipid and cholesterol concentrations were greatly reduced. The absence of endogenous bile acids did not change the hepatic clearance rate of a tracer dose of radiolabelled SGLC, but markedly decreased its biliary excretion rate. It was concluded that the hepatotoxic effect of SGLC is much more pronounced in rats with an exhausted bile acid pool, possibly due to a slower biliary excretion of the toxic compound. This phenomenon may have clinical implications for patients with a contracted bile acid pool.     

Ursodeoxycholic acid reduces lipid peroxidation and mucin secretagogue activity in gallbladder bile of patients with cholesterol gallstones

Background Recently it has been postulated that gallbladder mucin hypersecretion observed in the pathogenesis of cholesterol gallstone disease may be induced by biliary lipid peroxidation. Ursodeoxycholic acid treatment reduces mucin concentration and the formation of cholesterol crystals in the gallbladder bile of patients with cholesterol gallstones and this effect might be mediated by a decrease of biliary lipid peroxidation. Material and methods In a double‐blind, placebo‐controlled trial patients with symptomatic cholesterol gallstones received either ursodeoxycholic acid (750 mg daily) (n = 10) or placebo (n = 12) 10–12 days prior to cholecystectomy. As a marker for lipid peroxidation malondialdehyde was measured in bile together with mucin concentration. In addition, the mucin secretagogue activity of the individual bile samples was assessed in cultured dog gallbladder epithelial cells. Results Ursodeoxycholic acid therapy resulted in a significant reduction of lipid peroxidation in bile as determined by the biliary malondialdehyde concentration (1·36 ± 0·28 vs. 2·05 ± 0·38 µmol L^?1; P < 0·005) and the malondialdehyde (µmol L^?1)/total bile acid (mmol L^?1) ratio (0·02 ± 0·005 vs. 0·06 ± 0·01; P < 0·001). Furthermore, a decrease in mucin concentrations (0·7 ± 0·3 vs. 1·3 ± 0·5 mg mL^?1; P < 0·005) and of the mucin secretagogue activity of gallbladder bile (0·9 ± 0·2 vs. 2·2 ± 0·3 times control; P < 0·001) was observed. Conclusions The reduction of lipid peroxidation and mucin secretagogue activity of gallbladder bile induced by ursodeoxycholic acid treatment may contribute to the beneficial effects of this drug on gallbladder bile composition and symptoms in cholesterol gallstone patients.     

Bile Salt Sulphates in Cholestasis

Abstract. Bile salt sulphates were determined in serum and urine of 40 patients with severe cholestasis due to extrahepatic obstruction, hepatitis, cirrhosis and metastases of the fiver. Mono-, di- and tri-sulphates of bile salts were identified by column chromatography following intravenous administration of ¹⁴C-cholate. Quantitative analysis was done by gas-liquid chromatography following solvolysis. In our patients more than 50% of the bile salts excreted by the urine were sulphated (76.9% mono-sulphates, 21.3% di-sulphates, 1.8% tri-sulphates). In contrast less than 10% of serum bile salts were sulphated. Therefore the renal clearance of bile salt sulphates was more than 15 times greater than the clearance of non-sulphated bile salts. There were no significant differences in patients with extrahepatic obstruction, hepatitis, cirrhosis and metastases of the liver. – It is concluded that urinary excretion of mono- and di-sulphates of bile salts represents an important excretory mechanism in patients with cholestatic liver disease. In most patients only trace amounts of tri-sulphated bile salts were excreted in the urine.