Bile acids and their sulphated and glucuronidated derivatives in bile, plasma, and urine of children with intrahepatic cholestasis: effects of phenobarbital treatment

Abstract. Bile acids and their sulphated and glucuronidated derivatives were studied in three children with persistent intrahepatic cholestasis, two children with intrahepatic biliary hypoplasia, and four healthy children. In children with cholestasis, biliary bile acids consisted of 11(±0–3) % 3 β-hydroxy-delta-5-cholenoic acid, 2-1(± 0–6) % lithocholic acid, 2-2(± 11) % deoxy-cholic acid, 5–8(±2-2) % ursodeoxycholic acid, 39-1(± 1 -4) % chenodeoxycholic acid, 0–5(± 0 2) % hyo-cholic acid, and 49-3(± 3 0) % cholic acid. Of these bile acids 121 (±l 9) % were sulphated and 4–5 (±0 6) % were glucuronidated. In healthy children, biliary bile acids consisted of 0–7 (±0–4) % lithocholic acid, 3–4 (±0 8.) % deoxycholic acid, 0–1 (±0 1) % ursodeoxycholic acid, 32-7 (±6 9) % chenodeoxycholic acid, and 631 (±7 1) % cholic acid. Of these bile acids, 0–6±0 1 % were sulphated and 0–2 ±0 1% were glucuronidated (mean ± SEM). In the urine of healthy children, 3-3(±0 6) mg/24 h bile acids (1–5±0 3 mg sulphates and 0–1 ±0 1 mg glucuronides) were excreted, in the urine of children with cholestasis 61-4 (± 10 2) mg/24 h (30 2 ±7 1 mg sulphates and 5 6 ±1 2 mg glucuronides) were excreted. Thus in children with cholestasis the amounts of sulphated and glucuronidated bile acids are greater than in healthy controls. Substantial amounts of sulphated and glucuronidated bile acids are excreted in bile and urine of these patients. Phenobarbitone treatment in the five children with cholestasis led to a reduction of serum bile acids from 90 4 (± 13 2) μg/ml to 39 3(±3 6) μ//ml, a relative increase of bile acid glucuronides in bile from 45 (±0 6)% to 8 l(±0 6)%, a slight alteration of the bile acid sulphates in bile from 121(±l 9) % to 111 (± 1 2)% and no alteration of the bile acid spectrum. Urinary excretion of bile acids decreased from 61 4 (± 10 2) mg/24 h to 34 7(±3 0) mg/24 h. Phenobarbitone treatment of children with cholestasis thus induced glucuronidation of bile acids but had no significant effect on sulphation or on formation of individual bile acids.     

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.     

Identification and quantitative determination of urinary bile acids excreted in cholestasis

The monohydroxy bile acids, 3β-hydroxy-5-cholenoic acid and lithocholic acid and the dihydroxy bile acid, ursodeoxycholic acid have been identified by means of combined gas chromatography-mass spectrometry in urine of patients suffering from acute hepatitis, obstructive jaundice and intermittent jaundice, due to cholelithiasis. The occurrence of these bile acids in obstructive jaundice is suggested to be due to primary hepatic synthesis, since deoxycholic acid, the most sensitive indicator for the enterobacterial metabolism of bile acids, failed to be detected in significant quantities in the urine of these patients. The decrease of the content of deoxycholic acid in the urinary bile acid fraction seems to be of diagnostic value in recognition of complete obstruction. The total daily excretion of bile acids with the urine correlates with the degree of cholestasis, as could be judged from comparisons with serum bilirubin values. The occurrence of 3β-hydroxy-5-cholenoic acid seems to reflect an altered sterol metabolism in cholestasis.     

Determination of 3 beta-hydroxylated bile acids in human serum and liver tissue

3 beta, 7 alpha-Dihydroxy-5 beta-cholanoic acid (3 beta, 7 alpha-diOH) was detected in serum of 3 patients with intrahepatic cholestasis. 3 beta, 7 beta-dihydroxy-5 beta-cholanoic acid (3 beta, 7 beta-diOH) appeared in serum of those patients after treatment with ursodeoxycholic acid (UDC). These bile acids were also detected in only unconjugated fractions of serum of another 7 patients with chronic liver diseases, but not in liver tissue of them. The liver does not seem to from these bile acids itself because they were absent in liver tissue.     

Bile acids and progesterone metabolites in intrahepatic cholestasis of pregnancy

The pathogenesis of intrahepatic cholestasis of pregnancy (ICP) can be related to abnormalities in the metabolism and disposition of sex hormones and/or bile acids, determined by a genetic predisposition interacting with environmental factors. The total amount of oestrogens and progesterone circulating in the blood or excreted in the urine of ICP patients is similar to normal pregnancies. Thus, the search for the cause has been focused on abnormal hormone metabolites. The cholestatic potential of some D-ring oestrogen metabolites is supported by experimental and clinical data. Similar observations with regard to bile acids and progesterone metabolites are still scarce. This article reviews current knowledge in this field, including our own data. Bile acid synthesis appears to be reduced in patients with ICP, in whom primary conjugated bile acids are retained in blood. The major bile acid in blood and urine of these patients is cholic acid instead of chenodeoxycholic acid present in normal pregnancies. Hydroxylation and sulfation of bile acids are enhanced, while glucuronidation appears to be of lesser importance. The synthesis of progesterone appears unimpaired, while the profiles of progesterone metabolites in plasma and urine are different from normal pregnancies, with a larger proportion of mono- and disulfated metabolites, mainly 3alpha,5alpha isomers. Glucuronidated metabolites, however, are unchanged. With the administration of ursodeoxycholic acid (UDCA) to patients with ICP, pruritus and serum liver values are improved, the concentration of bile acids in blood is diminished and the proportion of their conjugated metabolites returned to normal. Simultaneously, the concentration of sulfated progesterone metabolites in blood and their urinary excretion are reduced. The serum levels of bile acids and progesterone metabolites before UDCA administration and their decrease during treatment do not correlate with each other. We propose that patients with ICP have a selective defect in the secretion of sulfated progesterone metabolites into bile and speculate that this may be caused by genetic polymorphism of canalicular transporter(s) for steroid sulfates or their regulation. Interaction with oestrogen metabolites and/or some exogenous compounds may further enhance the process triggering ICP in genetically predisposed individuals.     

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.     

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.     

Synthesis of 13C-labeled chenodeoxycholic, hyodeoxycholic, and ursodeoxycholic acids for the study of bile acid metabolism in liver disease.

In order to study the glycosidic conjugation of chenodeoxycholic, hyodeoxycholic, and ursodeoxycholic acids in patients with cholestasis after oral administration of pharmacological amounts of the respective bile acids avoiding the application of radioactive tracers we synthesized [24-13C]chenodeoxycholic, [24-13C]hyodeoxycholic, and [24-13C]ursodeoxycholic acids. The reaction intermediates of the bile acid syntheses were characterized by infrared spectroscopy. Purity was confirmed using thin-layer chromatography as well as gas chromatography-mass spectrometry. The 13C atom excess of approximately 90% of the synthesized bile acids was the same as the 13C atom excess of the sodium [13C]cyanide used for the labeling reaction confirming the successful synthesis. After oral administration of 0.5 g of [24-13C]ursodeoxycholic acid to a healthy volunteer, 13C label was detected in the nonamidated and glycine- or taurine conjugated glucosides and the N-acetylglucosaminide of ursodeoxycholic acid in urine. This establishes ursodeoxycholic acid as the first bile acid so far known to undergo both of the recently described glycosidic conjugation reactions in humans.     

Ursodeoxycholic acid improves ethinyl estradiol–induced cholestasis in the rat

Abstract. The effect of oral chronic administration of ursodeoxycholic acid has been examined in rats with cholestasis induced by ethinyl estradiol. Ursodeoxycholic acid at the dose of 25 mg kg^-1 per day during 4 days, did not improve the decrease in basal bile flow and bile acid secretion induced by ethinyl estradiol alone. In contrast, when ursodeoxycholic acid was given at the same dose during 10 days, basal bile flow was significantly improved and basal bile acid secretion was restored to control values. When ursodeoxycholic acid was given at the dose of 500 mg kg^-1 per day, basal bile flow and bile acid output were not further improved. However, bile flow and bile acid output under taurocholate infusion were restored to control values. Bile of rats treated with ursodeoxycholic acid was enriched with this bile acid. These results show a significant improvement of ethinyl estradiol-induced cholestasis in rats after chronic administration of ursodeoxycholic acid and support the use of this bile acid in intrahepatic cholestasis in man.     

Ursodeoxycholic acid treatment in humans: effects on plasma and biliary lipid metabolism with special reference to very low density lipoprotein triglyceride and bile acid kinetics

Ursodeoxycholic acid reduces biliary saturation with cholesterol and may induce dissolution of cholesterol gallstones in man. In order to characterize the effects of this potentially useful bile acid on plasma lipid metabolism, we determined lipoprotein levels and very low density lipoprotein (VLDL) triglyceride kinetics in six hypertriglyceridaemic and three normolipidaemic subjects before and after 4-6 weeks of ursodeoxycholic acid treatment at a daily dose of 15 mg kg-1 body weight. The plasma levels of low density lipoprotein (LDL), high density lipoprotein (HDL) and total cholesterol were not significantly affected by therapy. Nor were the plasma level and apparent formation of VLDL triglycerides changed. In five subjects, the effects of a low dose (7.5 mg kg-1 body weight day-1 for 4-6 weeks) of ursodeoxycholic acid on biliary lipid composition and kinetics of cholic acid and chenodeoxycholic acid were determined. The relative concentration of cholesterol in bile was reduced to the same level as during treatment with a high dose of ursodeoxycholic acid. The synthesis rates of bile acids were not suppressed with ursodeoxycholic acid. It is concluded that, unlike chenodeoxycholic acid, ursodeoxycholic acid does not suppress endogenous bile acid production. The efficiency at lower doses, and the lack of effects on plasma lipid metabolism, may make ursodeoxycholic acid a more attractive alternative for clinical attempts of gallstone dissolution.     

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.     

Separate transport systems for biliary secretion of sulfated and unsulfated bile acids in the rat.

Biliary secretion of 3 alpha-sulfated bile acids has been studied in Wistar rats with an autosomal recessive defect in the hepatic transport of bilirubin. Liver function, established by measurement of various enzymes in plasma, by enzyme histochemical methods, and by electron microscopy, appeared to be normal in these rats. Serum levels of unconjugated, monoglucuronidated, and diglucuronidated bilirubin were 0.62, 1.62, and 6.16 mumol/liter, respectively, compared with 0.17, 0.08, and 0.02 mumol/liter in control rats. Biliary bilirubin secretion was strongly reduced in the mutant animals: 0.21 +/- 0.03 vs. 0.39 +/- 0.03 nmol/min per 100 g body wt in control rats. Despite normal biliary bile acid output, bile flow was markedly impaired in the mutant animals, due to a 53% reduction of the bile acid-independent fraction of bile flow. The transport maximum for biliary secretion of dibromosulphthalein (DBSP) was also drastically reduced (-53%). Biliary secretion of intravenously administered trace amounts of the 3 alpha-sulfate esters of 14C-labeled taurocholic acid (-14%), taurochenodeoxycholic acid (-39%), taurolithocholic acid (-73%), and glycolithocholic acid (-91%) was impaired in the jaundiced rats compared with controls, in contrast to the biliary secretion of the unsulfated parent compounds. Hepatic uptake of sulfated glycolithocholic acid was not affected in the jaundiced animals. Preadministration of DBSP (15 mumol/100 g body wt) to normal Wistar rats significantly impaired the biliary secretion of sulfated glycolithocholic acid, but did not affect taurocholic acid secretion. We conclude that separate transport systems in the rat liver exist for biliary secretion of sulfated and unsulfated bile acids; the sulfates probably share secretory pathways with the organic anions bilirubin and DBSP. The described genetic defect in hepatic transport function is associated with a reduced capacity to secrete sulfated bile acids into bile; this becomes more pronounced with a decreasing number of hydroxyl groups on the sulfated bile acid's molecule.     

Effect of cholestyramine on bile acid pattern and synthesis during administration of ursodeoxycholic acid in man

BACKGROUND: Cholestyramine is the first-line treatment for cholestasis-induced pruritus and is prescribed along with ursodeoxycholic acid (UDCA) in patients with cholestatic liver diseases. Impairment of the intestinal absorption of endogenous hydrophobic bile acids by cholestyramine is well known. It is unclear, however, whether cholestyramine also impairs the absorption of the hydrophilic bile acid, UDCA, in man. AIMS: To study serum levels of UDCA and endogenous bile acids as well as endogenous bile acid synthesis during simultaneous or separate administration of UDCA and cholestyramine in vivo; and absorption of UDCA both in the presence and absence of its hydrophobic epimer, chenodeoxycholic acid (CDCA), by cholestyramine in vitro. PATIENTS AND METHODS: Five healthy subjects received UDCA (12.5 +/- 0.5 mg kg-1 daily) as a single dose for periods of 14 days with or without cholestyramine (4 g daily). Fasting serum levels of bile acids and of 7alpha-hydroxy-4-cholesten-3-one (alpha-HC), a measure of endogenous bile acid synthesis, were determined by gas chromatography and high pressure liquid chromatography, respectively. In vitro, bile acid solutions were incubated for 24 h in the presence or absence of cholestyramine, and bile acid concentrations were determined in the supernatant. RESULTS: Simultaneous administration of UDCA and cholestyramine in man led to a decrease of fasting serum levels of UDCA by 60% when compared to UDCA serum levels during administration of UDCA alone. In contrast, serum levels of endogenous bile acids were not affected and alpha-HC serum levels were found increased 2. 7-fold indicating stimulation of endogenous bile acid synthesis by cholestyramine. Administration of cholestyramine and UDCA at an interval of 5 h tended to diminish the effect of cholestyramine on UDCA serum levels. In vitro, conjugated and unconjugated UDCA were effectively bound by cholestyramine both in the presence and absence of hydrophobic bile acids. CONCLUSIONS: The results strongly support the recommendation to administer UDCA and cholestyramine at different times of day.     

Increased formation of ursodeoxycholic acid in patients treated with chenodeoxycholic acid

The formation of ursodeoxycholic acid, the 7 beta-hydroxy epimer of chenodeoxycholic acid, was investigated in three subjects with cerebrotendinous xanthomatosis and in four subjects with gallstones. Total biliary bile acid composition was analyzed by gas-liquid chromatography before and after 4 months of treatment with 0.75 g/day of chenodeoxycholic acid. Individual bile acids were identified by mass spectrometry. Before treatment, bile from cerebrotendinous xanthomatosis (CTX) subjects contained cholic acid, 85%; chenodeoxycholic acid, 7%; deoxycholic acid, 3%; allocholic acid, 3%; and unidentified steroids, 2%; while bile from gallstone subjects contained cholic acid, 45%; chenodeoxycholic acid, 43%; deoxycholic acid, 11%, and lithocholic acid, 1%. In all subjects, 4 months of chenodeoxycholic acid therapy increased the proportion of this bile acid to approximately 80% and decreased cholic acid to 3% of the total biliary bile acids, the remaining 17% of bile acids were identified as ursodeoxycholic acid. After the intravenous injection of [3H]chenodeoxycholic acid, the specific activity of biliary ursodeoxycholic acid exceeded the specific activity of chenodeoxycholic acid, and the resulting specific activity decay curves suggested precursor-product relationships. When [3H]7-ketolithocholic acid was administrated to another patient treated with chenodeoxycholic acid, radioactivity was detected in both the ursodeoxycholic acid and chenodeoxycholic acid fractions. These results indicate that substantial amounts of ursodeoxycholic acid are formed in patients treated with chenodeoxycholic acid. The ursodeoxycholic acid was synthesized from chenodeoxycholic acid presumably via 7-ketolithocholic acid.     

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.     

Glucuronides of unconjugated 6-hydroxylated bile acids in urine of a patient with malabsorption

The excretion of bile acids in urine from a patient with chronic malabsorption was investigated. Bile acids were separated according to mode of conjugation using a lipophilic anion exchanger, diethylaminohydroxypropyl Sephadex LH-20. Following hydrolysis, individual bile acids were analyzed by computerized GC/MS. In addition, bile acid glucuronides were isolated and their methyl ester trimethylsilyl ether derivatives were directly analyzed by GC/MS. The patient had a normal or slightly increased excretion of bile acids in urine. Bile acids carrying a hydroxyl group at C-6 constituted about 40% of the total. Tetrahydroxylated bile acids were present which have not been found in healthy subjects. Glucuronides of otherwise unconjugated bile acids accounted for 20% of the total. About 90% of these conjugates were 6-hydroxylated, hyodeoxycholic acid being the major bile acid. It is suggested that a specific abnormality of bile acid metabolism is related to the disease in this patient.     

Analysis of conjugated and unconjugated bile acids in serum and jejunal fluid of normal subjects

A reliable method is described for the determination of conjugated and unconjugated bile acids in serum and jejunal fluid. Bile acids are extracted using reverse-phase octadecylsilane bonded silica cartridges and are separated into their unconjugated and conjugated fractions using the lipophilic anion exchanger diethylaminohydroxypropyl Sephadex LH-20 (DEAP-LH-20). The conjugated fraction can be separated into a glycine and a taurine fraction, using the same anion exchanger. The bile acids are measured using a hydroxysteroid dehydrogenase-fluorimetric assay for serum and a hydroxysteroid dehydrogenase-photometric assay for jejunal fluid. The normal fasting serum value of total 3 alpha-hydroxy bile acids amounts to 3.5 +/- 2.8 mumol/l (mean +/- SD, range 1.4-10.8, n = 22). The corresponding unconjugated bile acid fraction amounts to 39.9 +/- 11.2% (range 20.7-64.6%) of total bile acids. The concentration of conjugated bile acids became significantly elevated 30, and 60 min after a standard meal, whereas that of unconjugated bile acids remained unchanged. In jejunal fluid only conjugated bile acids are found, as well in fasting subjects as postprandial, 30 or 60 min after a standard meal.     

Serum unconjugated bile acids in patients with small bowel bacterial overgrowth

Concentrations of total and unconjugated bile acids in serum were measured fasting and 2 h postprandially in 9 patients with a positive [14C]glycocholate breath test consistent with small bowel bacterial overgrowth and in 13 controls. Gas-liquid chromatography-mass spectrometry (GLC-MS) and enzymatic-fluorometric assays were both used. In contrast to previous work, total serum bile acids were only occasionally elevated in patients with bacterial overgrowth. Total 2 h postprandial unconjugated bile acids, however, were elevated in 7/9 patients when measured by GLC-MS and in 6/9 when measured by the enzymatic-fluorometric method. The best separation between patients and controls was achieved by GLC-MS determinations of 2 h postprandial unconjugated cholic acid or primary bile acids, which were abnormal in 8/9 patients. This study indicates that measurement of serum bile acids may be a useful approach to the diagnosis of bacterial overgrowth, but would require accessible methods for separating and measuring cholic acid or unconjugated primary bile acids in post-prandial sera.     

Serum unconjugated bile acids: qualitative and quantitative profiles in ileal resection and bacterial overgrowth

Qualitative and quantitative profiles of unconjugated bile acids in the serum obtained over a 24-h period from three patients with ileal resections and one with a bacterial overgrowth are described. Unconjugated serum bile acids were determined using the high sensitivity and resolution of capillary column gas liquid chromatography after their rapid extraction and isolation using reverse phase octadecylsilane bonded silica cartridges and the lipophilic gel Lipidex 1000. Unconjugated serum bile acid concentrations were elevated throughout the day in both ileum resected patients and in conditions involving bacterial overgrowth when compared to healthy subjects. Total conjugated cholic acid concentrations were expectedly low in both intestinal disorders and were without the postprandial increases generally observed in healthy subjects. Qualitative gas chromatographic profiles of serum unconjugated bile acids in bacterial overgrowth distinctly revealed a predominance of deoxycholic acid and other secondary bile acids in all samples, while, in conditions of an impaired enterohepatic circulation, deoxycholic acid was absent or present in only trace amounts. The potential significance of measuring serum unconjugated bile acids in intestinal disorders is discussed.     

Effect of ageing on postprandial conjugated and unconjugated serum bile acid levels in healthy subjects

Abstract. Colorectal cancer is a disease of elderly subjects. A decreased ileal absorption of bile acids in elderly subjects may lead to an increased exposure of the colonic mucosa to secondary bile acids. This may contribute to an enhanced risk of colorectal cancer. In this study fasting and postprandial conjugated and unconjugated serum levels of cholic, chenodeoxycho-lic, and deoxycholic acid in 12 elderly and 12 younger subjects were investigated. Intestinal transit time, gallbladder emptying and jejunal bacterial flora were also studied in both age groups. Fasting levels of conjugated and unconjugated serum bile acids were similar in both age groups. Postprandial levels of all individual conjugated bile acids increased to a significantly higher extent in the younger subjects. Postprandial unconjugated serum bile acid levels did not differ significantly between both age groups, although unconjugated deoxycholic levels tended to increase to higher levels in the elderly. Results of jejunal bacterial counts, gallbladder emptying and intestinal transit time were similar in both groups. These data suggest that conjugated bile acids are reabsorbed less effectively in elderly subjects.