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.     

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 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 pattern in human amniotic fluid

Individual bile acids were determined in twenty-nine amniotic fluid specimens obtained from twenty-six women between the 32nd and 41st week of gestation. Total bile acid concentration ranged from 0.4 to 4.8 mumol/l with a mean of 1.57 mumol/l. Besides the two major bile acids of man, cholic acid and chenodeoxycholic acid, 3beta-hydroxy-5-cholenoic acid was found in all, lithocholic acid in ten and deoxycholic acid in nine of the twenty-nine amniotic fluid samples. 3beta-Hydroxy-5-cholenoic acid averaged 39.8% of total bile acids during 32-37 weeks of gestation and 20.2% at term (P less than 0.01). These findings point towards important differences between fetal and adult bile metabolism and may reflect maturation of hepatic bile acid biosynthesis near term.     

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.     

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.     

Delta 4-3-oxosteroid 5 beta-reductase deficiency described in identical twins with neonatal hepatitis. A new inborn error in bile acid synthesis.

A new inborn error in bile acid synthesis, manifest in identical infant twins as severe intrahepatic cholestasis, is described involving the delta 4-3-oxosteroid 5 beta-reductase catalyzed conversion of the key intermediates, 7 alpha-hydroxy-4-cholesten-3-one and 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one for chenodeoxycholic and cholic acid synthesis, to the respective 3 alpha-hydroxy-5 beta (H) products. This defect was detected by fast atom bombardment ionization-mass spectrometry from an elevated excretion and predominance of taurine conjugated unsaturated hydroxy-oxo-bile acids. Gas chromatography-mass spectrometry confirmed these to be 7 alpha-hydroxy-3-oxo-4-cholenoic and 7 alpha,12 alpha-dihydroxy-3-oxo-4-cholenoic acids (75-92% of total). Fasting serum bile acid concentrations were greater than 37 mumol/liter; chenodeoxycholic acid was the major bile acid, but significant amounts of allo(5 alpha-H)-bile acids (approximately 30%) were present. Biliary bile acid concentration was less than 2 mumol/liter and consisted of chenodeoxycholic, allo-chenodeoxycholic, and allo-cholic acids. These biochemical findings, which were identical in both infants, indicate a defect in bile acid synthesis involving the conversion of the delta 4-3-oxo-C27 intermediates into the corresponding 3 alpha-hydroxy-5 beta(H)-structures, a reaction that is catalyzed by a delta 4-3-oxosteroid-5 beta reductase enzyme. This defect resulted in markedly reduced primary bile acid synthesis and concomitant accumulation of delta 4-3-oxo-and allo-bile acids. These findings indicate a pathway in bile acid synthesis whereby side chain oxidation can occur despite incomplete alterations to the steroid nucleus, and lend support for an active delta 4-3-oxosteroid 5 alpha-reductase catalyzing the conversion of the delta 4-3-oxosteroid intermediates to the respective 3 alpha-hydroxy-5 alpha(H)-structures.     

The identification of urinary bile alcohols by gas chromatography–mass spectrometry in patients with liver disease and in healthy individuals

Abstract. The neutral steroid fractions in the urine of eleven patients suffering from various forms of liver disease with cholestasis and of ten healthy individuals were studied by glass capillary gas chromatography-mass spectrometry. The steroid conjugates in urine were enzymatically solvolysed, the liberated steroids extracted and transformed into the trimethylsilylether for measurements.
The excretion rates of androstane and pregnane metabolites of patients with liver disease were far lower than those of healthy persons. The main compounds in the urine of the former were the bile alcohols 27 - nor -3α, 7α, 12α, 24, 25 - pentahydroxy - 5β - cholestane and 3α, 7α, 12α, 25, 26 - pentahydroxy - 5β - cholestane. Our data suggest a correlation between the excretion rates of these bile alcohols and the serum levels of bilirubin. While the excretion rate of the two bile alcohols in the urine of healthy individuals was approximately 0.24 mg/24 h (0.6 μmol/24 h) a patient with a serum bilirubin of 841 μmol/1 excreted 4 mg/24 h (9 μmol/24 h). The accumulation of bile alcohols described in this study possibly indicates alternative pathways of cholic acid formation in liver disease.     

Metabolism of Mono-and Dihydroxylated Bile Acids in Preparations of Human Liver

Abstract. The metabolism of tritium-labelled taurodeoxycholic acid, taurochenodeoxycholic acid, taurolithocholic acid and of [24-¹⁴C] lithocholic acid and [24-¹⁴C] 3-keto-5β-cholanoic acid was studied in fractions of human liver homogenates.—In the presence of microsomal fraction fortified with an NADPH-generating system a small conversion of taurodeoxycholic acid into taurocholic acid occurred. No significant hydroxylation of taurochenodeoxycholic acid, taurolithocholic acid or free lithocholic acid could be detected. In the presence of microsomal fraction fortified with NAD lithocholic acid was converted into 3-keto-5β-cholanoic acid. In the presence of microsomal fraction fortified with NADH, 3-keto-5β-cholanoic acid was reduced to isolithocholic acid and lithocholic acid (ratio 3:1). In the presence of 100000 × g supernatant fraction only lithocholic acid was formed from 3-keto-5β-cholanoic acid.—The physiological significance of the ability of human liver to convert lithocholic acid into isolithocholic acid is discussed.     

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.     

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.     

Faecal bile acid analysis and intestinal absorption in Crohn''s disease before and after ileal resection

Abstract. Faecal bile acids were analysed by gas chromatography in 104 patients. Total bile acids exceeded 1.5 mmol/24 h in 33% of forty-five unoperated patients and in 90% of those having undergone an ileal resection. Lithocholic and deoxycholic fractions were lower in the unoperated patients than in the control group ( P < 0.05 and P < 0.005) and much lower after ileal resection than in unoperated patients ( P < 0.001). A significant correlation ( r = 0.58; P < 0.001) was found between total bile acids and relative proportions of primary bile acids in operated patients, untreated by antibiotics or sulfasalazine. Dihydroxy bile acids (predominantly chenodeoxycholic acid) correlated with faecal weight in unoperated patients ( r = 0.47, P < 0.01) and in 0–50 cm ( r = 0.69, P < 0.001) and 50–100 cm ( r = 0–63, P < 0.01) ileal resection groups. Our results suggest that the frequently altered bile acid composition is related to a shortening of colonic transit time which reduces the exposure of primary bile acids to bacterial 7α-dehydroxylase.     

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.     

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.     

Serum bile acid composition in patients with cystic fibrosis

Serum bile acid composition was examined in detail using capillary column gas chromatography and mass spectrometry in 10 children with cystic fibrosis (CF) and 4 healthy children. The mean total bile acid concentration in fasting serum of CF patients was 2.33 +/- 0.84 mumol/l, slightly lower than but not statistically significantly different from healthy controls (mean 2.86 +/- 0.98 mumol/l) and appeared to show no relationship to the degree of exocrine pancreatic insufficiency. Analysis of individual serum bile acids in these children showed that cholic acid represented less than 10% of the total bile acids. Chenodeoxycholic acid was the predominant serum bile acid; the mean concentration in CF patients was 0.98 +/- 0.51 mumol/l, lower than for the healthy controls (1.69 +/- 0.84 mumol/l). Concentrations of lithocholic acid, 3 beta-hydroxy-5-cholenoic, ursodeoxycholic and 3 beta, 7 alpha, 12 alpha-trihydroxy-5 beta-cholanoic acids in fasting serum samples of the CF patients were not significantly different from the healthy control sera but were higher than those normally found in adults. Measurements of fecal bile acid excretion indicated an increased loss of primary bile acids in patients with CF consistent with an impairment of the enterohepatic circulation of bile acids.     

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

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

ISOLATED FECAL MICROORGANISMS CAPABLE OF 7 α-DEHYDROXYLATING BILE ACIDS

Strains of microorganisms capable of 7α-dehydroxylation of chenodeoxycholate were isolated from rat and human feces. All the strains were strictly anaerobic, non-motile, moderately themioresistant Gram-positive rods. They showed some saccharolytic properties with the production of both acid and gas. They were H₂S-positive but indole-, skatole-, citrate-, catalase-, and oxidase-negative. The isolated strains capable of 7α-dehydroxylation of chenodeoxycholate were also able to oxidize the hydroxyl groups at C-3 and C-7 to keto groups. The following metabolites were isolated: 3-keto-7α-hydroxy-5β-cholanoic acid, 3α-hydroxy-7-keto-5β-cholanoic acid, 3α-hydroxy-5β-cholanoic acid, and 3-keto-5β-cholanoic acid. The isolated strains did not have the enzymes necessary for hydrolyzing conjugated bile acids. In mixed anaerobic cultures of fecal microorganisms, extensive reduction of the 3-keto group to the 3β-hydroxyl group occurred. The microorganism(s) responsible for this reaction have as yet not been isolated.     

Capillary gas chromatographic determinations of urinary bile acids and bile alcohols in CTX patients proving the ineffectivity of ursodeoxycholic acid treatment

Urine samples and serum samples of a patient with cerebrotendinous xanthomatosis (CTX) were investigated by means of capillary gas chromatography, both before and during oral treatment with ursodeoxycholic acid (UDCA), and the results compared with those obtained during chenodeoxycholic acid (CDCA) therapy. The predominantly excreted bile alcohol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol and two abnormal bile acids, i.e. 23-norcholic acid and 23-hydroxycholic acid were determined. In addition, the serum cholestanol/cholesterol ratio was determined. Whereas previous experiments demonstrated that the urinary excretion of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol and the abnormal bile acids decreased within a few weeks during CDCA therapy, the present study shows that their urinary excretions remain essentially the same during UDCA treatment. In contrast to the decrease in the serum cholestanol/cholesterol ratio during CDCA therapy, this ratio remains essentially the same during UDCA therapy. It is therefore concluded that, in contrast to CDCA therapy, UDCA treatment is not effective in the treatment of CTX.     

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.     

Bile acid inhibition of interferon activity in human lymphocytes: no evidence of oxidative stress

Cholestasis and bile acids are two factors involved in resistance to interferon therapy in patients with chronic hepatitis C. As bile acids inhibit the biological activity of this cytokine in vitro and are capable of generating oxidative stress in hepatocytes, we investigated the potential involvement of such a mechanism in human lymphocytes. Thus, we evaluated (a) the effects of bile acids (0–200 μmol L⁻¹) on lymphocyte reduced glutathione content and malondialdehyde production and (b) the ability of antioxidants to prevent the inhibitory effect of chenodeoxycholic acid on interferon-induced lymphocyte 2',5'-oligoadenylate synthetase activity, an index of the biological activity of interferon. We found that treatment of lymphocytes with bile acids for 24 h did not induce malondialdehyde release or significantly modify cellular reduced glutathione content. Synthetic precursors of glutathione ( N -acetylcysteine and S -adenosylmethionine) and antioxidants (superoxide dismutase and catalase) had no preventive influence on the inhibitory effect of chenodeoxycholic acid on interferon-induced 2',5'-oligoadenylate synthetase activity. These negative results do not provide evidence for the use of glutathione precursors in cholestatic conditions associated with viral diseases.