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.     

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.     

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.     

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.     

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 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.     

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.     

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.     

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.     

Urinary bile acids during development of recurrent cholestasis of pregnancy

The pregnancies of two patients with mild intrahepatic cholestasis of pregnancy (RCP) were followed with detailed analyses of bile acids in urine. About twenty-five different bile acids were determined by GC/MS following separation according to mode of conjugation. The results were collated with the clinical course of the disease. The first detectable change in bile acid excretion was the appearance of tetrahydroxylated bile acids at about the 30th gestational week. Somewhat later and concomitant with the rise in urinary oestriol, the total bile acid excretion started to increase. In one of the patients, who had a maximum total excretion of 84 mumol/24 h, deoxycholic acid was a major constituent, comprising about 40% of the total. The same patient had only slightly elevated levels of tetrahydroxylated bile acids and serum amino-transferases. The possible effect of low-fat diet on these results is discussed. Monohydroxylated bile acids were present throughout the pregnancies in small amounts and their role as aetiological factors is discussed. The care of RCP patients is outlined, and the need for simple, specific and quantitative methods for following the course of RCP is pointed out.     

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.     

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.     

Biliary bile acids in uncomplicated pregnancy and in cholestasis of pregnancy

Conjugated and sulfated bile acids were determined by gas-liquid chromatography and by high-pressure liquid chromatography in gallbladder bile samples of four pregnant women at term and of two patients with cholestasis of pregnancy. In healthy pregnant women the mean ratio of cholyl/chenodeoxycholyl/deoxycholyl glycine was 3.7 : 1 : 0.23 and that of taurine conjugates 3.0 : 1 : 0.25, respectively. In gallbladder bile pool of non-pregnant females these ratios were 1.0 : 1 : 0.00 and 1.0 1 : 0.70, respectively. Thus cholic acid predominated in pregnancy bile. In patients with cholestasis of pregnancy, cholid acid comprised 90% of total biliary bile acids, the proportion of chenodeoxycholic acid was greatly decreased and nonsulfated deoxycholic acid was not detected. The proportion of sulfated bile acids of total biliary bile acids was between 0.4 and 1.2% in uncomplicated pregnancy and 0.3 and 0.5% in cholestasis patients.     

Enhancement of the urinary excretion of non-sulphated and sulphated radioactive bile acids by sodium acetate in the bile duct obstructed rat

The renal clearances of [14C]glycocholate, [14C]taurocholate and [3H]glycochenodeoxycholate-3-sulphate were determined in bile duct obstructed rats. Comparisons of the bile acid clearances with glomerular filtration rates (GFR) indicate that most of the filtered bile acids are reabsorbed. Inhibition studies with p-aminohippurate (PAH) and probenecid suggest that a proportion of the bile acids in urine is secreted. Attempts were made to increase the renal clearance of the bile acids by the administration of pharmacological agents. An infusion of sodium acetate (0.3 mol/l) increased the clearance of the radioactive bile acids and augmented the urinary excretion of endogenous 3 alpha-hydroxy bile acids and reduced their concentration in plasma.     

An enzymatic method of determining 7 alpha-hydroxylated bile acids in urine

A recently developed enzymatic method of determining unsulphated 7 alpha-hydroxylated bile acids in serum was evaluated for the analysis of bile acids in urine after hydrolysis of the sulphate esters. Results obtained using the enzymatic method were compared to those obtained by radioimmunoassay of 7 alpha-hydroxylated bile acids in urine samples from patients. Results of the enzymatic method were also compared to results of gas-liquid chromatography of individual bile acids after group separation of the bile acids on Sephadex-DEAP-LH-20. There was significant correlation between the enzymatic method and radioimmunoassay for bile acid determination in urine. The analysis of different conjugates after group separation of bile acids revealed no unspecific reactions when compared to gas-liquid chromatography. The reference range for 7 alpha-hydroxylated urinary bile acids was in agreement with earlier published results.     

Bile acid excretion: the alternate pathway in the hamster.

The quantitative significance of renal excretion of bile acid ester sulfates as an alternate excretory pathway was evaluated in hamsters. After bile duct ligation, total serum bile acid fell from a mean level of 454 microgram/ml at 24 h to 64 microgram/ml by 96 h. During this period the bulk of the bile acid pool could be accounted for as esterified bile acids in urine. Renal pedicle ligation of animals with bile duct obstruction led to retention of the bile acid ester sulfates in serum. Thioacetamide hepatotoxicity diminished ester sulfation of bile acids causing diminished renal secretion with relatively greater retention of nonesterified bile acids in serum. We conclude that secretion of esterified bile acids by the kidney is an efficient alternate pathway for maintaining bile acid excretion in obstructive biliary tract disease. Coexistent hepatocellular disease diminishes ester sulfation and the effectiveness of the alternate pathway in maintaining bile acid excretion.     

Cholestasis induced by sodium taurolithocholate in isolated hamster liver

The mechanism of cholestasis (decreased bile flow) induced by taurolithocholate in the isolated perfused hamster liver was investigated. Taurocholate was infused to maintain bile acid output, and sulfobromophthalein (BSP) was administered to establish a BSP transport maximum in bile. The effects of taurolithocholate on bile flow and on the biliary secretion of BSP and bile acid anions were determined.A significant dose-response correlation was found between taurolithocholate and the degree of cholestasis. No significant hepatic morphologic alterations were observed. At low doses, cholestasis was reversible. A multiple regression equation was developed to validate the steroid dehydrogenase determination of total bile acids in bile that contained BSP. During cholestasis, output of bile acid was maintained by a significantly increased concentration of bile acid. Hepatic removal rate and transport maximum of BSP were significantly decreased, whereas BSP concentration, conjugation, and hepatic content were unaffected. The concentrating capacity for BSP in bile appeared to be the rate-limiting factor in BSP transport.Individual bile acids were determined by gas-liquid chromatography. Of the injected taurolithocholate, 40-50% was recovered in bile as lithocholic acid, 30% was converted to chenodeoxycholic acid, and only traces of lithocholic acid were detected in the perfusate after 4 hr. Cholic and chenodeoxycholic acids comprised 75-89%, and lithocholic acid comprised 11-25% of bile acids in bile after taurolithocholate injection; only traces of deoxycholic acid were seen. Small amounts of taurolithocholate sulfate were detected in bile by thinlayer chromatography. The outputs of sodium and potassium in bile were significantly diminished during cholestasis.A substantial fraction (75%) of basal bile flow in the isolated hamster liver was estimated to be independent of bile acid secretion. Cholestasis occurred after taurolithocholate, whereas bile acid secretion was maintained. The results indicate that the most likely mechanism for acute cholestasis induced by taurolithocholate in isolated hamster liver was interference with the bile acid-independent fraction of canalicular or ductular bile flow or both.     

Tetrahydroxylated bile acids in healthy human newborns

Urine was collected in four healthy infants, born after 35-39 weeks of gestation. The collections were made for 24 h starting immediately after birth. Bile acids were extracted, separated into conjugate groups, solvolysed, hydrolysed and quantified by gas-liquid chromatography as methyl ester trimethylsilyl ether derivatives. Identification was made by gas-liquid chromatography-mass spectrometry. Cholic acid was the predominating primary bile acid. Non-sulphated tetrahydroxylated bile acids, tentatively identified as 1,3,7,12- and 3,6,7,12-tetrahydroxycholanoic acids, were present in almost the same amounts as cholic acid. A previously unknown tetrahydroxylated cholanoic acid, which might be 2-hydroxylated hyocholic acid, was found in all infants in similar amounts. 3 beta-Hydroxy-5-cholenoic acid was present in the sulphate fraction in the urine from all infants. Lithocholic acid was not found. Small amounts of allo and 6-hydroxylated bile acids were mainly found in the sulphate fraction. Bile acids hydroxylated in the 1-position were found predominantly in the taurine fraction.     

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.