Determination of 3 beta-hydroxy-5-cholenoic acid in serum of hepatobiliary diseases--its glucuronidated and sulfated conjugates

3 beta-Hydroxy-5-cholenoic acid in the serum of control subjects and 62 patients with various hepatobiliary diseases was quantitated by mass fragmentography after separation into nonglucuronidated-nonsulfated, glucuronidated, and sulfated fractions. Deuterium-labeled deoxycholic acid and its glucuronide and sulfate were used as internal standards. Mean concentrations of total 3 beta-hydroxy-5-cholenoic acid in serum (mumole/liter) were as follows: Control subjects (14), 0.184; obstructive jaundice (15), 6.783; liver cirrhosis, compensated (12), 0.433, and decompensated (12), 1.636; chronic hepatitis (12), 0.241; and acute hepatitis (11), 2.364. Most of the 3 beta-hydroxy-5-cholenoic acid was glucuronidated or sulfated. Only in patients with obstructive jaundice did glucuronidation (60 +/- 14%) exceed sulfation (31 +/- 14%), sulfation exceeding glucuronidation in the others. The UDP-glucuronyltransferase might have different substrate specificities for 3 beta-hydroxy-5-cholenoic acid and other common bile acids, especially in the cholestatic state.     

Bile acid synthesis: down-regulation by monohydroxy bile acids

The regulation of bile acid synthesis was studied in rabbits after interruption of the enterohepatic circulation by choledochoureteral anastomosis. Total daily bile acid output was 772 +/- 130 (SD) mumol/24 h, of which greater than 95% was glycocholic acid. Administration of deoxycholic or cholic acid or their conjugates (300-800 mumol) or gall-bladder bile failed to down-regulate endogenous bile acid synthesis. In contrast, chenodeoxycholic acid administration did down-regulate bile acid synthesis, but this effect was related to the formation and excretion of lithocholic acid. This observation was confirmed by the finding that i.v. infusion of 10-20 mumol of either lithocholic acid or 3 beta-hydroxy-5-cholenoic acid significantly reduced cholic acid synthesis. Thus monohydroxy bile acids, derived from either hepatic or intestinal sources, participate in the down-regulation of bile acid synthesis.     

Serum concentrations of glucuronidated and sulfated bile acids in children with cholestasis

Serum concentrations of nonglucuronidated-nonsulfated, glucuronidated, and sulfated bile acids in 9 control children and 16 children with cholestasis were quantitated by mass fragmentography. Total bile acid levels in control children were 19.55 +/- 2.78 mumol/liter (mean +/- SEM), and glucuronidated and sulfated bile acids comprised 2.6 +/- 0.5 and 17 +/- 3.1%, respectively. In 9 patients with congenital biliary atrasia, total bile acid levels were 167.34 +/- 11.18 mumole/liter of which 2.1 +/- 0.3% were glucuronidated and 15 +/- 1.4% were sulfated. Lithocholic and 3 beta-hydroxy-5-cholenoic acids, which have hepatotoxic effects, were presented in only small amounts in cholestatic children, and they were almost all glucuronidated or sulfated. The percentages of glucuronidated bile acids in control and cholestatic children were lower than in healthy and cholestatic adults, which may be explained by the lower activity of UDP-glucuronyltransferase in neonatal liver.     

Bioconversion of 3beta-hydroxy-5-cholenoic acid into chenodeoxycholic acid by rat brain enzyme systems

We have previously demonstrated that the rat brain contains three unconjugated bile acids, and chenodeoxycholic acid (CDCA) is the most abundantly present in a tight protein binding form. The ratio of CDCA to the other acids in rat brain tissue was significantly higher than the ratio in the peripheral blood, indicating a contribution from either a specific uptake mechanism or a biosynthetic pathway for CDCA in rat brain. In this study, we have demonstrated the existence of an enzymatic activity that converts 3beta-hydroxy-5-cholenoic acid into CDCA in rat brain tissue. To distinguish marked compounds from endogenous related compounds, 18O-labeled 3beta-hydroxy-5-cholenoic acid, 3beta,7alpha-dihydroxy-5-cholenoic acid, and 7alpha-hydroxy-3-oxo-4-cholenoic acid were synthesized as substrates for in vitro incubation studies. The results clearly suggest that 3beta-hydroxy-5-cholenoic acid was converted to 3beta,7alpha-dihydroxy-5-cholenoic acid by microsomal enzymes. The 7alpha-hydroxy-3-oxo-4-cholenoic acid was produced from 3beta,7alpha-dihydroxy-5-cholenoic acid by the action of microsomal enzymes, and Delta4-3-oxo acid was converted to CDCA by cytosolic enzymes. These findings indicate the presence of an enzymatic activity that converts 3beta-hydroxy-5-cholenoic acid into CDCA in rat brain tissue. Furthermore, this synthetic pathway for CDCA may relate to the function of 24S-hydroxycholesterol, which plays an important role in cholesterol homeostasis in the body.     

Bile acid synthesis in cell culture

Confluent cultures of Hep G2 cells were found to synthesize chenodeoxycholic and cholic acids continually. Chenodeoxycholic acid was synthesized at the rate of 58 +/- 8.6 micrograms/96 h, a rate more than 7-fold greater than that for cholic acid. Addition of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol but not the -3 alpha, 7 alpha-diol was followed by an increase in cholic acid synthesis, thus indicating a relatively low 12 alpha-hydroxylase activity. Endogenous synthesis of monohydroxy bile acid ester sulfates was found, with maximum rates of 135 and 74 micrograms/96 h for lithocholic and 3 alpha-hydroxy-5-cholenoic acids, respectively. Incubation of Hep G2 cells in medium containing 25% D2O permitted a comparison of the precursor/product relationship of cholesterol with 3 beta-hydroxy-5-cholenoic acid. The pattern of incorporation of deuterium was in accordance with that expected, thus allowing the conclusion that this monohydroxy bile acid is derived from cholesterol and should be considered together with chenodeoxycholic and cholic acids as a primary bile acid.     

Radioimmunoassay of urinary 3 beta-hydroxy-5-cholenoyl glycine in hepatobiliary disease

A radioimmunoassay for 3 beta-hydroxy-5-cholenoyl glycine in human urine has been developed. The antiserum was elicited with the antigen in which the steroid hapten is linked to a bovine serum albumin through the C-19 position. The [125I]-tyrosine derivative of the hapten was used as radioligand. The standard curves were linear ranging from 10 to 320 ng/mL. The cross-reactivities with other bile acids were not detectable and below 0.3% with cholesterol. Sample preparation includes extraction of 3 beta-hydroxy-5-cholenoyl glycine from urine and solvolysis of the sulfates--main form present in urine. Urinary excretion of 3 beta-hydroxy-5-cholenoyl glycine was 0.373 +/- 0.133 mumol/day in healthy adults. Urinary excretion of 3 beta-hydroxy-5-cholenoyl glycine increased in chronic liver dysfunction, hepatoma and obstructive jaundice in this order.     

Effect of bile duct ligation on bile acid and cholesterol metabolism in rats

The effects of bile duct ligation on bile acid and cholesterol metabolism were examined in male Wistar strain rats. Quantitative and qualitative changes of bile acids and cholesterol in serum and urine occurred; beta-muricholic acid predominantly increased in serum and urine and the ratio of urinary cholic acid and beta-muricholic acid changed from about 5:3 on day 1 to about 1:8 on day 5 under biliary obstruction. The form of the increased urinary bile acids was mainly taurine-conjugated and partly sulfated. Under conditions of bile duct ligation on day 5, 14C-labeled 3 beta-hydroxy-5-cholenoic, lithocholic, and chenodeoxycholic acids were intragastrically administered to the rats after pretreatment with antibiotics and the metabolites of these three acids were investigated. 3 beta-Hydroxy-5-cholenoic acid was most efficiently converted to beta-muricholic acid. The present study strongly suggested the presence of an alternative metabolic pathway induced by bile duct ligation, which caused the change in composition of urinary bile acids, and especially the marked increase in beta-muricholic acid formation. A possible alternative pathway for bile acid biosynthesis under biliary obstruction in rats is postulated.     

Bile acid metabolism in early life: studies of amniotic fluid

Bile acid metabolism of the human fetus was examined in early gestation (weeks 13-19) and compared with the full-term fetus from the analysis of amniotic fluid collected from healthy pregnant women. Total individual bile acids were determined by gas-liquid chromatography-mass spectrometry after solvolysis and hydrolysis of bile acid conjugates. Additionally, bile acids were separated according to their mode of conjugation by lipophilic anion exchange chromatography. Qualitatively the bile acid profiles of amniotic fluid in early gestation were similar and markedly different from those of full-term fetuses. Chenodeoxycholic acid was the major bile acid identified in early gestation and concentrations exceeded those of cholic acid, but by full term this relationship was reversed. Over 50 bile acids were identified in the amniotic fluids, these included C-1, C-4, and C-6 hydroxylated species and reflected primary hepatic synthesis by the fetus. At full term, 7 alpha,12 alpha-dihydroxy-3-oxo-4-cholenoic acid was one of the major bile acids identified in amniotic fluid. The monohydroxy bile acids lithocholic and 3 beta-hydroxy-5-cholenoic acids were present in significant proportions during early gestation, but by full term these accounted for only a few percent of the total bile acids. Quantitatively the total bile acid concentration of amniotic fluid was less than 4 mumol/l. The majority of bile acids were found to be glyco-, tauro-, and sulfate-conjugates. The more hydrophobic bile acids tended to be preferentially sulfated. These data indicate that significant and major changes in bile acid metabolism take place between early and late gestation in the human fetus.     

Cytosolic ferritin and lipid-associated ferritin are metabolically different in guinea-pig livers.

1. Using a human hepatoma (Hep G2) cell line that continually synthesizes 3 beta-hydroxy-5-cholenoic acid, lithocholic acid, chenodeoxycholic acid and cholic acid we have determined the metabolism and biological effects of 26-hydroxycholesterol and 7 alpha-hydroxycholesterol. 2. Addition of 26-hydroxycholesterol to the medium (6 microM) downregulated cholesterol and chenodeoxycholic acid synthesis. 3. The predominant metabolite of 26-hydroxycholesterol was 3 beta-hydroxy-5-cholenoic acid. 4. Cholesterol synthesis was not affected by the addition of 7 alpha-hydroxycholesterol (6 and 12 microM). The predominant metabolite of 7 alpha-hydroxycholesterol was chenodeoxycholic acid. 5. In Hep G2 cells 7 alpha-hydroxylation of 26-hydroxycholesterol is not well expressed.     

Bile acid synthesis. Metabolism of 3 beta-hydroxy-5-cholenoic acid to chenodeoxycholic acid

Metabolism of 3 beta-hydroxy-5-cholenoic acid to chenodeoxycholic acid has been found to occur in rabbits and humans, species that cannot 7 alpha-hydroxylate lithocholic acid. This novel pathway for chenodeoxycholic acid synthesis from 3 beta-hydroxy-5-cholenoic acid led to a reinvestigation of the pathway for chenodeoxycholic acid from 3 beta-hydroxy-5-cholenoic acid in the hamster. Simultaneous infusion of equimolar [1,2-3H]lithocholic acid and 3 beta-hydroxy-5-[14C]cholenoic acid indicated that the 14C enrichment of chenodeoxycholic acid was much greater than that of lithocholic acid. Thus, in all these species, a novel 7 alpha-hydroxylation pathway exists that prevents the deleterious biologic effects of 3 beta-hydroxy-5-cholenoic acid.     

Occurrence of 3 beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid as normal constituents in human blood

Three unconjugated C27 bile acids were found in plasma from healthy humans. They were isolated by liquid-solid extraction and anion-exchange chromatography and were identified by gas-liquid chromatography-mass spectrometry, microchemical reactions, and ultraviolet spectroscopy as 3 beta-hydroxy-5-cholestenoic, 3 beta,7 alpha-dihydroxy-5-cholestenoic, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acids. Their levels often exceeded those of the unconjugated C24 bile acids and the variations between individuals were smaller than for the C24 acids. The concentrations in plasma from 11 healthy subjects were 67.2 +/- 27.9 ng/ml (mean +/- SD) for 3 beta-hydroxy-5-cholestenoic acid, 38.9 +/- 25.6 ng/ml for 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 81.7 +/- 27.9 ng/ml for 7 alpha-hydroxy-3-oxo-4-cholestenoic acid. The levels of the individual acids were positively correlated to each other and not to the levels of the C24 acids. The cholestenoic acids were below the detection limit (20-50 ng/ml) in bile and C27 bile acids present in bile were not detected in plasma.     

Bile acid synthesis in rat liver peroxisomes: metabolism of 26-hydroxycholesterol to 3 beta-hydroxy-5-cholenoic acid

Rat liver peroxisomes have been found to oxidize 26-hydroxycholesterol, the product of cholesterol C-26 hydroxylation to 3 beta-hydroxy-5-cholenoic acid. Peroxisomes were purified by differential and equilibrium density centrifugation in a steep linear metrizamide gradient to greater than 95% purity. Purity of peroxisomes was determined by measurement of specific marker enzymes. The activities of cytochrome oxidase (a mitochondrial marker) and acid phosphatase (a lysosomal marker) in the purified peroxisome fractions were below the level of detection. Esterase activity indicated a 2-4% microsomal contamination. Subsequent to incubation of peroxisomes with [16,22-3H]-26-hydroxycholesterol, the reaction products were extracted, methylated, acetylated, and subjected to thin-layer, high pressure liquid, and gas-liquid chromatographic analyses. 3 beta-Hydroxy-5-cholenoic acid was the major identifiable metabolite of 26-hydroxycholesterol. Incubations of pure microsomal fractions (greater than 99%) with 26-hydroxycholesterol under the same conditions demonstrated that the production of 3 beta-hydroxy-5-cholenoic acid by peroxisomes was not attributable to microsomal contamination. This study demonstrates that peroxisomes participate in the side-chain oxidation of intermediates in bile acid synthesis.     

Human cecal bile acids: concentration and spectrum

To obtain information on the concentration and spectrum of bile acids in human cecal content, samples were obtained from 19 persons who had died an unnatural death from causes such as trauma, homicide, suicide, or drug overdose. Bile acid concentration was measured via an enzymatic assay for 3alpha-hydroxy bile acids; bile acid classes were determined by electrospray ionization mass spectrometry and individual bile acids by gas chromatography mass spectrometry and liquid chromatography mass spectrometry. The 3alpha-hydroxy bile acid concentration (mumol bile acid/ml cecal content) was 0.4 +/- 0.2 mM (mean +/- SD); the total 3-hydroxy bile acid concentration was 0.6 +/- 0.3 mM. The aqueous concentration of bile acids (supernatant after centrifugation) was identical, indicating that most bile acids were in solution. By liquid chromatography mass spectrometry, bile acids were mostly in unconjugated form (90 +/- 9%, mean +/- SD); sulfated, nonamidated bile acids were 7 +/- 5%, and nonsulfated amidated bile acids (glycine or taurine conjugates) were 3 +/- 7%. By gas chromatography mass spectrometry, 10 bile acids were identified: deoxycholic (34 +/- 16%), lithocholic (26 +/- 10%), and ursodeoxycholic (6 +/- 9), as well as their primary bile acid precursors cholic (6 +/- 9%) and chenodeoxycholic acid (7 +/- 8%). In addition, 3beta-hydroxy derivatives of some or all of these bile acids were present and averaged 27 +/- 18% of total bile acids, indicating that 3beta-hydroxy bile acids are normal constituents of cecal content. In the human cecum, deconjugation and dehydroxylation of bile acids are nearly complete, resulting in most bile acids being in unconjugated form at submicellar and subsecretory concentrations.     

Bile acid and sterol solubilization in 2-hydroxypropyl-beta-cyclodextrin.

The use of 2-hydroxypropyl-beta-cyclodextrin has made it possible to prepare stable aqueous solutions of cholesterol, 26-hydroxycholesterol, 7 alpha-hydroxycholesterol, and monohydroxy bile acids such as lithocholic and 3 beta-hydroxy-5-cholenoic acids. These solutions are suitable for cell culture studies and for parenteral administration to animals.     

Serum selenium concentration related to myocardial infarction and fatty acid content of serum lipids.

A longitudinal case-control study of 33 patients with one or more risk factors for coronary heart disease and 64 controls showed that the serum selenium concentration (range 0.63-1.33 mumol/l (50-105 micrograms/l] was not associated with development of clinical manifestations of coronary heart disease during a follow up of five to seven years. The content of polyunsaturated fatty acids, especially eicosapentaenoic acid, in serum cholesterol esters and phospholipids was positively correlated with selenium concentration. As a low content of polyunsaturated fatty acids in serum lipids was an independent risk factor for coronary heart disease in these subjects it may be hypothesised that the high coronary risk in subjects with a very low serum selenium concentration (less than 0.57 mumol/l (less than 45 micrograms/l] might be due not to selenium deficiency but to the coexisting low concentrations of polyunsaturated fatty acids in serum.     

Presence of monohydroxy bile acids in the urinary precipitates: a pitfall in the analysis of urinary bile acids

The importance of the method of handling the urinary precipitates frequently present in urine samples, especially after freezing-thawing, for bile acid analysis is emphasized because of the presence of a considerable proportion of monohydroxy bile acids such as lithocholic and 3 beta-hydroxy-5-cholenoic acids. Filtration of the urinary precipitates may lead to the underestimation of these important bile acid species.     

Concurrent occurrence of 3 beta,12 alpha-dihydroxy-5-cholenoic acid associated with 3 beta-hydroxy-5-cholenoic acid and their preferential urinary excretion in liver diseases

3 beta-Hydroxy-(delta 5-3 beta-ol), 3 beta,12 alpha-dihydroxy-(delta 5-3 beta,12 alpha-ol), 3 beta,7 alpha-dihydroxy-(delta 5-3 beta,7 alpha-ol) and 3 beta,7 beta-dihydroxy-(delta 5-3 beta,7 beta-ol) 5-cholenoic acids were identified in patients with liver diseases by gas-liquid chromatography-mass spectrometry (GLC-MS). Of these unusual 3 beta-hydroxy-5-en-metabolites, delta 5-3 beta-ol and delta 5-3 beta,12 alpha-ol were found as major components in the urine of patients with liver diseases (cholestasis, liver cirrhosis, chronic hepatitis, acute hepatitis). Other 3 beta-dihydroxy-5-en-metabolites, delta 5-3 beta,7 alpha-ol and delta 5-3 beta,7 beta-ol, were found as minor components in the urine. The levels of delta 5-3 beta-ol and delta 5-3 beta,12 alpha-ol in urine were correlated with their levels in serum, with total bile acids in the urine, and with liver function, implying that the degree of their increment correlated well with the severity of liver diseases. The most abundant amounts of delta 5-3 beta-ol and delta 5-3 beta,12 alpha-ol were found in the urine as sulfate conjugates in comparison with bile, portal and hepatic venous sera, and liver tissue of the patients. The biliary excretion and hepatic extraction of these 3 beta-hydroxy-5-en-unsaturated bile acids were more impaired and inefficient than those of cholic and chenodeoxycholic acids.     

Bile acids of patients with renal failure receiving chronic hemodialysis

Bile acids in serum, urine and dialysate of 8 patients with renal failure in chronic hemodialysis were analyzed by gas chromatography and gas chromatography-mass spectrometry. The following results were obtained: 1. Lithocholic acid, 3 beta-hydroxy-5-cholen-24-oic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, and cholic acid were identified in hemodialysate as well as in serum and urine. 2. The serum bile acid concentration of the patients was 2.78 +/- 0.57 micrograms/mL before hemodialysis and 1.34 +/- 0.48 micrograms/mL after a 5-h period hemodialysis with cuprophane membrane. The proportions of secondary bile acids in predialysis and postdialysis serum of patients were significantly higher than those of healthy subjects. 3. Two out of 8 patients excreted urine. But the amounts of bile acids in urine of the patients were very small compared to those of healthy subjects. 4. The amount of bile acids removed from blood by hemodialysis was 0.70 +/- 0.25 mg. In dialysate, cholic acid constituted a larger proportion of the total bile acids, and lithocholic acid a smaller proportion, when compared to those in urine of patients and healthy subjects.     

Hydrogenation alternatives: effects of trans fatty acids and stearic acid versus linoleic acid on serum lipids and lipoproteins in humans.

The objective of this study was to compare the effects of linoleic acid (cis,cis-C18:2(n-6)) and its hydrogenation products elaidic (trans-C18:1(n-9)) and stearic acid (C18:0) on serum lipoprotein levels in humans. Twenty-six men and 30 women, all normolipemic and apparently healthy, completed the trial. Three experimental diets were supplied to every subject for 3 weeks each, in random order (multiple cross-over). The Linoleate-diet provided 12.0% of total energy intake as linoleic acid, 2.8% as stearic acid, and 0.1% as trans fatty acids. The Stearate-diet supplied 3.9 energy % as linoleic acid, 11.8% stearic acid, and 0.3% trans fatty acids. The Trans-diet provided 3.8 energy % as linoleic acid, 3.0% stearic acid, and 7.7% as monounsaturated trans fatty acids, largely elaidic acid (trans-C18:1(n-9)). Other nutrients were constant. Fasting blood was sampled at the end of each dietary period. Mean (+/- SD) serum LDL cholesterol was 109 +/- 24 mg/dl (2.83 +/- 0.63 mmol/l) on the Linoleate-diet. It rose to 116 +/- 27 mg/dl (3.00 +/- 0.71 mmol/l) on the Stearate-diet (change, 7 mg/dl or 0.17 mmol/l, P = 0.0008) and to 119 +/- 25 mg/dl (3.07 +/- 0.65 mmol/l) on the Trans-diet (change, 9 mg/dl or 0.24 mmol/l, P less than 0.0001). High density lipoprotein (HDL) cholesterol decreased by 2 mg/dl (0.06 mmol/l, P less than 0.0001) on the Stearate-diet and by 4 mg/dl (0.10 mmol/l, P less than 0.0001) on the Trans-diet, both relative to linoleic acid. Our findings show that 7.7% of energy (mean, 24 g/day) of trans fatty acids in the diet significantly lowered HDL cholesterol and raised LDL cholesterol relative to linoleic acid. Combination with earlier results (Mensink, R. P., and M. B. Katan. 1990. N. Engl. J. Med. 323: 439-445) suggests a linear dose-response relation. Replacement of linoleic acid by stearic acid also caused somewhat lower HDL cholesterol and higher LDL cholesterol levels. Hydrogenation of linoleic acid to either stearic or trans fatty acids produces fatty acids that may increase LDL and decrease HDL cholesterol relative to linoleic acid itself.     

Clostridium scindens baiCD and baiH genes encode stereo-specific 7alpha/7beta-hydroxy-3-oxo-delta4-cholenoic acid oxidoreductases

Secondary bile acids, formed by intestinal bacteria, are suggested to play a significant role in cancers of the gastrointestinal tract in humans. Bile acid 7alpha/beta-dehydroxylation is carried out by a few species of intestinal clostridia which harbor a multi-gene bile acid inducible (bai) operon. Several genes encoding enzymes in this pathway have been cloned and characterized. However, no gene product(s) has yet been assigned to the production of 3-oxo-Delta4-cholenoic acid intermediates of cholic acid (CA), chenodeoxycholic acid (CDCA) or ursodeoxycholic acid (UDCA). We previously reported that the baiH gene encodes an NADH:flavin oxidoreductase (NADH:FOR); however, the role of this protein in bile acid 7-dehydroxylation is unclear. Homology searches and secondary structural alignments suggest this protein to be similar to flavoproteins which reduce alpha/beta-unsaturated carbonyl compounds. The baiH gene product was expressed in Escherichia coli, purified and discovered to be a stereo-specific NAD(H)-dependent 7beta-hydroxy-3-oxo-Delta4-cholenoic acid oxidoreductase. Additionally, high sequence similarity between the baiH and baiCD gene products suggests the baiCD gene may encode a 3-oxo-Delta4-cholenoic acid oxidoreductase specific for CDCA and CA. We tested this hypothesis using cell extracts prepared from E. coli overexpressing the baiCD gene and discovered that it encodes a stereo-specific NAD(H)-dependent 7alpha-hydroxy-3-oxo-Delta4-cholenoic acid oxidoreductase.