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.     

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.     

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.     

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.     

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.     

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.     

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.     

Urinary excretion of tetrahydroxylated bile acids in children with alpha 1-antitrypsin deficiency and neonatal cholestasis

Urinary excretion of bile acids was investigated in seven infants with alpha 1-antitrypsin deficiency and neonatal cholestasis. The infants were followed prospectively and the bile acid analysis was repeated after 2-8 years in four of the patients. Bile acid excretion during 24 h was quantified by gas-liquid chromatography and the bile acids were identified by gas-liquid chromatography-mass spectrometry. In contrast to healthy controls, all the patients excreted tetrahydroxylated bile acids during cholestasis and in the postcholestatic period. The patients who developed cirrhosis during the observation period had lower concentration of tetrahydroxylated bile acids than those with a more favourable course. Our findings suggest that tetrahydroxylation of bile acids might be an alternative pathway for elimination of bile acids in cholestasis of infancy, and that those infants exhibiting a good capacity of polyhydroxylation might have a better prognosis.     

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 of a new inborn error in bile acid synthesis: mutation of the oxysterol 7alpha-hydroxylase gene causes severe neonatal liver disease.

We describe a metabolic defect in bile acid synthesis involving a deficiency in 7alpha-hydroxylation due to a mutation in the gene for the microsomal oxysterol 7alpha-hydroxylase enzyme, active in the acidic pathway for bile acid synthesis. The defect, identified in a 10-wk-old boy presenting with severe cholestasis, cirrhosis, and liver synthetic failure, was established by fast atom bombardment ionization-mass spectrometry, which revealed elevated urinary bile acid excretion, a mass spectrum with intense ions at m/z 453 and m/z 510 corresponding to sulfate and glycosulfate conjugates of unsaturated monohydroxy-cholenoic acids, and an absence of primary bile acids. Gas chromatography-mass spectrometric analysis confirmed the major products of hepatic synthesis to be 3beta-hydroxy-5-cholenoic and 3beta-hydroxy-5-cholestenoic acids, which accounted for 96% of the total serum bile acids. Levels of 27-hydroxycholesterol were > 4,500 times normal. The biochemical findings were consistent with a deficiency in 7alpha-hydroxylation, leading to the accumulation of hepatotoxic unsaturated monohydroxy bile acids. Hepatic microsomal oxysterol 7alpha-hydroxylase activity was undetectable in the patient. Gene analysis revealed a cytosine to thymidine transition mutation in exon 5 that converts an arginine codon at position 388 to a stop codon. The truncated protein was inactive when expressed in 293 cells. These findings indicate the quantitative importance of the acidic pathway in early life in humans and define a further inborn error in bile acid synthesis as a metabolic cause of severe cholestatic liver disease.     

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.     

Influence of portacaval shunt on bile formation in the rat

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

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.     

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.     

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.     

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.     

On the homogeneity of stools with respect to bile acid composition and normal day-to-day variations: a detailed qualitative and quantitative study using capillary column gas chromatography-mass spectrometry

Fecal bile acid excretion was determined using recently developed techniques in order to investigate: (a) the extent of the homogeneity in composition and concentration of individual bile acids in a single stool sample, (b) the detailed qualitative and quantitative day-to-day variations in total and individual bile acids in the typical healthy adult, (c) information on the relative proportions of conjugated bile acids in healthy stools, and (d) inter-individual variations in fecal bile acid excretion. Bile acids were extracted from feces and separated into groups based upon their mode of conjugation using lipophilic gel chromatography, prior to analysis by capillary column gas chromatography-mass spectrometry. The majority of bile acids were excreted in the unconjugated form, while in all samples, conjugated bile acids accounted for < 6% of the total fecal bile acids excreted, of which sulphated bile acids represented < 3% of the total. Quantitative total and individual bile acid excretion, determined from single daily collections exhibited wide variations in values from day-to-day, and in accordance with early findings, indicates the need to use a minimum of 3- to 5-day collections for a more reliable index of bile acid excretion in feces. Examination of frozen and sectioned single stools revealed wide variations in water content and in quantitative bile acid concentration and composition within the stool. These data indicate random stool samples, which are commonly used in clinical studies, and data expressed as concentrations to be unsatisfactory for the accurate determination of fecal bile acid excretion.     

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.     

Inhibition of secondary bile acid formation in the large intestine by lactulose in healthy subjects of two different age groups

Secondary bile acids have been implicated in colonic carcinogenesis. Transformation of primary into secondary bile acids (7 alpha-dehydroxylation) in the large bowel is a pH-dependent process. Inhibition of this reaction could be achieved by lowering colonic pH. We, therefore, studied the effects of lactulose (a non-absorbable disaccharide), which is capable of acidifying colonic contents, on secondary bile acid metabolism. Because this metabolism is age dependent, lactulose was given (0.3 g kg-1 twice daily for 12 weeks) to nine middle-aged (age 31-54 years; mean 45.7) and ten elderly subjects (age 56-81 years; mean 66.4). Twice before, and after 6 and 12 weeks' lactulose administration, biliary and faecal bile acids, whole gut transit time, faecal weight and dry weight, and faecal pH were recorded. The concentration of (iso)lithocholic and deoxycholic acid in faeces was higher in elderly subjects (P less than 0.05) but the excretion was comparable. After lactulose the concentration and excretion of the major secondary bile acids decreased. The primary bile acid fraction rose from 5% before, to more than 20% after, lactulose (P less than 0.05). Faecal weight increased and faecal dry weight decreased, resulting in a higher faecal water output during lactulose. Whole gut transit time did not change. The faecal pH dropped after 6 (P less than 0.05) and further after 12 weeks' lactulose (P less than 0.05). The percentage deoxycholic acid in bile was higher, and cholic acid lower, in elderly subjects (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)