On the origin of the cholestenoic acids in human circulation

3 Beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid are metabolites of cholesterol present at significant concentrations (40-80 ng/ml) in human circulation. The 7 alpha-hydroxylated acids may be formed from cholesterol via two major pathways initiated by oxidations at either the 7 alpha- or 27-positions. In an attempt to clarify the origin and possible precursor-product relationships between these cholestenoic acids, we measured their deuterium enrichment in a unique experiment, after infusion of 10 g of [2H(6)]-cholesterol to a healthy volunteer. The observed extent and time-course of deuterium enrichment of circulating 3 beta-hydroxy-5-cholestenoic and 3 beta,7 alpha-dihydroxy-5-cholestenoic acid were almost identical, while different from that of cholesterol and 7 alpha-hydroxycholesterol. Notably, the deuterium enrichment of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid was similar to that of 7 alpha-hydroxycholesterol (and its metabolite 7 alpha-hydroxy-4-cholesten-3-one), though distinct from the other cholestenoic acids. Finally, the enrichment of unesterified 27-hydroxycholesterol followed a similar, though less pronounced, time curve to the delta(5)-cholestenoic acids. In conclusion, these results suggest that plasma 3 beta-hydroxy-5-cholestenoic acid is formed from a pool of cholesterol distinct from that used for the formation of the bulk of 27-hydroxycholesterol. The results are also in accordance with a formation of 3 beta,7 alpha-dihydroxy-5-cholestenoic acid directly from 3 beta-hydroxy-5-cholestenoic acid, and a formation of most of the circulating 7 alpha-hydroxy-4-cholesten-3-one from 7 alpha-hydroxycholesterol. These results are consistent with a flux of 7 alpha-hydroxycholesterol from the liver into the circulation, and an extrahepatic metabolism of this steroid into 7 alpha-hydroxy-3-oxo-4-cholestenoic acid.     

Concentrations of cholestenoic acids in plasma from patients with reduced intestinal reabsorption of bile acids

The concentrations of 3 beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid were determined in plasma from patients treated with cholestyramine or subjected to resection of the ileum or colon. The values were compared with those for conjugated and unconjugated C24 bile acids. Patients with an intact ileum but without colon had normal levels of cholestenoic acids. Patients treated with cholestyramine or with ileal resection had elevated levels of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid (median values 189 and 233 ng/ml, respectively, compared to 85 ng/ml in controls). The levels of the other two C27 acids were normal in cholestyramine-treated and low in ileoresected patients and were positively correlated to each other but not to the 3-oxo-delta 4 acid. There were no consistent correlations between the levels of C27 acids and those of conjugated or unconjugated C24 bile acids. The results indicate an increased formation of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid in subjects having a stimulated activity of cholesterol 7 alpha-hydroxylase.     

Bile acid synthesis in cultured human hepatoblastoma cells.

Biosynthetic pathways to bile acids have been studied in HepG2 cells, a well-differentiated human hepatoblastoma cell line. Cholesterol metabolites, in total 29, were isolated from culture media and cells by liquid-solid extraction and anion-exchange chromatography and were identified by gas-liquid chromatography-mass spectrometry. The production rates/concentrations of cholic acid (CA) and chenodeoxycholic acid (CDCA) in media from control cells were 71 and 74 ng/10(7) cells/h, respectively. Major bile acid precursors were 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA), 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholestenoic acid, 7 alpha-hydroxy-3-oxo-4-cholenoic acid, and 7 alpha, 12 alpha-dihydroxy-3-oxo-5 beta-cholanoic acid, their concentrations being 60, 30, 23, and 10 ng/10(7) cells/h, respectively. These and nine other isolated intermediates formed essentially complete metabolic sequences from cholesterol to CA and CDCA. The remaining steroids were metabolites of the intermediates or autooxidation products of cholesterol. These findings and the observed effect of dexamethasone on production rates suggest that in HepG2 cells the major biosynthetic pathways to primary bile acids start with 7 alpha-hydroxylation of cholesterol and oxidation to 7 alpha-hydroxy-4-cholesten-3-one followed by hydroxylation at either the 26 or 12 alpha position. CDCA is formed by the sequence of 26-hydroxylation, oxidation, and degradation of the side chain and A-ring reduction. CA is formed by the sequence of 12 alpha-hydroxylation, 26-hydroxylation, oxidation, and degradation of the side chain and reduction of the A-ring. An alternative pathway to CA included A-ring reduction of the intermediate 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholestenoic acid to form THCA prior to side chain cleavage. These pathways are not limited to HepG2 cells but may also be important in humans.     

Correlation between plasma levels of 7alpha-hydroxy-4-cholesten-3-one and cholesterol 7alpha-hydroxylation rates in vivo in hyperlipidemic patients

BACKGROUND/AIM: Hepatic bile acid synthesis is the main mechanism whereby the organism can degrade cholesterol. Plasma levels of 7alpha-hydroxy-4-cholesten-3-one have been reported to reflect bile acid synthesis and the expression or activity of the limiting enzyme of the main biosynthetic pathway, cholesterol 7alpha-hydroxylase. Aim of this study was to correlate the levels of this metabolite with the rates of cholesterol 7alpha-hydroxylation in vivo, a direct measurement of bile acid synthesis, in hyperlipidemic patients. DESIGN: Concentrations of 7alpha-hydroxy-4-cholesten-3-one were assayed by gas-liquid chromatography: mass spectrometry in plasma samples obtained in 18 patients with primary hyperlipoproteinemia who previously underwent determination of cholesterol 7alpha-hydroxylation rates in vivo by tritium release analysis. Both determinations were performed in basal conditions and after treatment with hypolipidemic drugs (the fibric acid derivatives gemfibrozil and bezafibrate, cholestyramine alone or associated with simvastatin). RESULTS: Changes in plasma 7alpha-hydroxy-4-cholesten-3-one profile closely reflected in vivo cholesterol 7alpha-hydroxylation rates during treatment with fibrates, cholestyramine and cholestyramine plus simvastatin. When plotting determinations from all studies (n=40), a very strict correlation was disclosed between plasma 7alpha-hydroxy-4-cholesten-3-one and cholesterol 7alpha-hydroxylation rates (r=0.81, P<0.001). CONCLUSIONS: Plasma 7alpha-hydroxy-4-cholesten-3-one closely mirrors measurements of cholesterol 7alpha-hydroxylation rates in vivo in hyperlipidemic subjects and therefore stands as a reliable marker of global bile acid synthesis. In view of the correlation observed, these data may help to interpret changes of plasma levels of this metabolite in terms of cholesterol balance quantification.     

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.     

Mechanism of intestinal 7 alpha-dehydroxylation of cholic acid: evidence that allo-deoxycholic acid is an inducible side-product

We previously reported that the 7 alpha-dehydroxylation of cholic acid appears to be carried out by a multi-step pathway in intestinal anaerobic bacteria both in vitro and in vivo. The pathway is hypothesized to involve an initial oxidation of the 3 alpha-hydroxy group and the introduction of a double bond at C4-C5 generating a 3-oxo-4-cholenoic bile acid intermediate. The loss of water generates a 3-oxo-4,6-choldienoic bile acid which is reduced (three steps) yielding deoxycholic acid. We synthesized, in radiolabel, the following putative bile acid intermediates of this pathway 7 alpha,12 alpha-dihydroxy-3-oxo-4-cholenoic acid, 7 alpha,12 alpha-dihydroxy-3-oxo-5 beta-cholanoic acid, 12 alpha-dihydroxy-3-oxo-4,6-choldienoic acid, and 12 alpha-hydroxy-3-oxo-4-cholenoic acid and showed that they could be converted to 3 alpha,12 alpha-dihydroxy-5 beta-cholanoic acid (deoxycholic acid) by whole cells or cell extracts of Eubacterium sp. VPI 12708. During studies of this pathway, we discovered the accumulation of two unidentified bile acid intermediates formed from cholic acid. These bile acids were purified by thin-layer chromatography and identified by gas-liquid chromatography-mass spectrometry as 12 alpha-hydroxy-3-oxo-5 alpha-cholanoic acid and 3 alpha,12 alpha-dihydroxy-5 alpha-cholanoic (allo-deoxycholic acid). Allo-deoxycholic acid was formed only in cell extracts prepared from bacteria induced by cholic acid, suggesting that their formation may be a branch of the cholic acid 7 alpha-dehydroxylation pathway in this bacterium.     

Evidence for a lack of regulatory importance of the 12 alpha-hydroxylase in formation of bile acids in man: an in vivo study

The possibility that the 12 alpha-hydroxylase involved in formation of bile acids is of regulatory importance for the ratio between cholic acid and chenodeoxycholic acid in bile was studied with an in vivo technique. [4-14C]7 alpha-Hydroxy-4-cholesten-3-one and [6 beta-3H]7 alpha, 12 alpha-dihydroxy-4-cholesten-3-one were synthesized, and a mixture of these two bile acid intermediates was administered intravenously in five healthy subjects and in one patient with severe liver cirrhosis. The patient with liver cirrhosis was included in the study because of a considerable reduction in biosynthesis of cholic acid. Since the [4-14C]-labeled steroid is an intermediate just proximal to and since the [6 beta-3H]-labeled steroid is an intermediate just distal to the 12 alpha-hydroxylase step, the 3H/14C ratio in the cholic acid formed should reflect the relative 12 alpha-hydroxylase activity. The 3H/14C ratio varied between 1.8 and 3.9 in the cholic acid isolated from the healthy subjects and was 3.6 in the cholic acid isolated from the patient with liver cirrhosis. The ratio between cholic acid and chenodeoxycholic acid varied between 0.6 and 3.9 in the bile from the control subjects and was only 0.4 in the bile from patients with liver cirrhosis. There was no correlation between the 3H/14C ratios and the ratios between cholic acid and chenodeoxycholic acid in bile.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholesterol is converted to 7 alpha-hydroxy-3-oxo-4-cholestenoic acid in liver mitochondria. Evidence for a mitochondrial sterol 7 alpha-hydroxylase.

The metabolism of cholesterol in isolated intact pig liver mitochondria has been investigated. Six major cholesterol metabolites were identified by gas-liquid chromatography-mass spectrometry, the metabolic end product being 7 alpha-hydroxy-3-oxo-4-cholestenoic acid. Incubations with the synthesized intermediates suggested that the major pathway from cholesterol to this acid proceeds via the sequence of 26-hydroxylation, 7 alpha-hydroxylation, further oxidation of the side chain and oxidation/isomerization in the A-ring. The observed reactions prove that in addition to a sterol 26-hydroxylase, pig liver mitochondria contain significant amounts of a 7 alpha-hydroxylase active on side chain oxygenated 3 beta-hydroxy-delta 5-C27 steroids, an oxidoreductase active in the side chain of 26-hydroxylated steroids and a 3 beta-hydroxy-delta 5 steroid oxidoreductase active on 7 alpha-hydroxylated C27 steroids. Since 7 alpha-hydroxy-3-oxo-4-cholestenoic acid is believed to be an important precursor of chenodeoxycholic acid, this study shows that the first reactions in the biosynthesis of bile acids can be exclusively mitochondrial and thereby bypass microsomal cholesterol 7 alpha-hydroxylase as the rate-limiting enzyme.     

Novel LC-MS/MS method for assay of 7alpha-hydroxy-4-cholesten-3-one in human plasma. Evidence for a significant extrahepatic metabolism

A new isotope dilution LC-MS/MS method for assay of 7alpha-hydroxy-4-cholesten-3-one without need for derivatization is described. This method was used in catheterization experiments on healthy fasting volunteers. The levels of this generally used marker for bile acid synthesis were slightly but significantly higher in the hepatic vein than in the brachial artery. In contrast, the levels of the precursor to 7alpha-hydroxy-4 cholesten-3-one, 7alpha-hydroxycholesterol, were the same in the two vessels. It is concluded that there is a net extrahepatic metabolism of 7alpha-hydroxy-4-cholesten-3-one. The similarity and very high correlation between the levels in the two vessels (r=0.97) are consistent with the contention that 7alpha-hydroxy-4-cholesten-3-one is a suitable marker for the activity of the hepatic cholesterol 7alpha-hydroxylase and thus bile acid synthesis.     

The degradation of cholic acid by Pseudomonas sp. N.C.I.B. 10590 under anaerobic conditions.

The bacterial degradation of cholic acid under anaerobic conditions by Pseudomonas sp. N.C.I.B. 10590 was studied. The major unsaturated neutral compound was identified as 12 beta-hydroxyandrosta-4,6-diene-3,17-dione, and the major unsaturated acidic metabolite was identified as 12 alpha-hydroxy-3-oxochola-4,6-dien-24-oic acid. Eight minor unsaturated metabolites were isolated and evidence is given for the following structures: 12 alpha-hydroxyandrosta-4,6-diene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-4,6-dien-3-one, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-1,4,6-trien-3-one, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-1,4,6-trien-3-one, 12 alpha-hydroxyandrosta-1,4-diene-3,17-dione, 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione, 3,12-dioxochola-4,6-dien-24-oic acid and 12 alpha-hydroxy-3-oxopregna-4,6-diene-20-carboxylic acid. In addition, a major saturated neutral compound was isolated and identified as 3 beta,12 beta-dihydroxy-5 beta-androstan-17-one, and the only saturated acidic metabolite was 7 alpha,12 alpha-dihydroxy-3-oxo-5 beta-cholan-24-oic acid. Nine minor saturated neutral compounds were also isolated, and evidence is presented for the following structures: 12 beta-hydroxy-5 beta-androstane-3,17-dione, 12 alpha-hydroxy-5 beta-androstane-3,17-dione, 3 beta,12 alpha-dihydroxy-5 beta-androstan-17-one, 3 alpha,12 beta-androstan-17-one, 3 alpha,12 alpha-dihydroxy-5 beta-androstan-17-one, 5 beta-androstane-3 beta,12 beta,17 beta-triol, 5 beta-androstane-3 beta,12 alpha,17 beta-triol, 5 beta-androstane-3 alpha,12 beta,17 beta-triol and 5 beta-androstane-3 alpha,12 alpha,17 beta-triol. The induction of 7 alpha-dehydroxylase and 12 alpha-dehydroxylase enzymes is discussed, together with the significance of dehydrogenation and ring fission under anaerobic conditions.     

Bile acids. LXXIII. Synthesis of analogs of 7 alpha-hydroxy-4-cholesten-3-one as substrates for hepatic steroid 12 alpha-hydroxylase

Analogs of 7 alpha-hydroxy-4-cholesten-3-one were prepared to ascertain structural features necessary for maximal activity of hepatic microsomal 12 alpha-steroid hydroxylase. Methyl 3 alpha,7 alpha-dihydroxy-5 beta-cholane-24-carboxylate derived from chenodeoxycholic acid was oxidized at C-3 with silver carbonate/Celite. The product was hydrolyzed and dehydrogenated with SeO2 to provide 3-oxo-7 alpha-hydroxy-4-cholene-24-carboxylic acid. 5 beta-Cholestane-3 alpha,7 alpha,25-triol and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol were similarly oxidized at C-3 and dehydrogenated to provide 7 alpha,25-dihydroxy-4-cholesten-3-one and 7 alpha,12 alpha,25-trihydroxy-4-cholesten-3-one, respectively. The products were characterized by thin-layer and gas chromatography, ultraviolet, infrared, proton resonance and mass spectrometry.     

Selective inhibition of CYP27A1 and of chenodeoxycholic acid synthesis in cholestatic hamster liver

The aim of this study was to explore the regulation of serum cholic acid (CA)/chenodeoxycholic acid (CDCA) ratio in cholestatic hamster induced by ligation of the common bile duct for 48 h. The serum concentration of total bile acids and CA/CDCA ratio were significantly elevated, and the serum proportion of unconjugated bile acids to total bile acids was reduced in the cholestatic hamster similar to that in patients with obstructive jaundice. The hepatic CA/CDCA ratio increased from 3.6 to 11.0 (P<0.05) along with a 2.9-fold elevation in CA concentration (P<0.05) while the CDCA level remained unchanged. The hepatic mRNA and protein level as well as microsomal activity of the cholesterol 7alpha-hydroxylase, 7alpha-hydroxy-4-cholesten-3-one 12alpha-hydroxylase and 5beta-cholestane-3alpha,7alpha,12alpha-triol 25-hydroxylase were not significantly affected in cholestatic hamsters. In contrast, the mitochondrial activity and enzyme mass of the sterol 27-hydroxylase were significantly reduced, while its mRNA levels remained normal in bile duct-ligated hamster. In conclusion, bile acid biosynthetic pathway via mitochondrial sterol 27-hydroxylase was preferentially inhibited in bile duct-ligated hamsters. The suppression of CYP27A1 is, at least in part, responsible for the relative decreased production of CDCA and increased CA/CDCA ratio in the liver, bile and serum of cholestatic hamsters.     

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.     

Biosynthesis of cholestanol from bile acid intermediates in the rabbit and the rat

Biliary 7 alpha-hydroxy-4-cholesten-3-one (an intermediate in bile acid biosynthesis) may be 7 alpha-dehydroxylated in the gut and further metabolized to cholestanol (Skrede, S., and Bj?rkhem, I. (1982) J. Biol. Chem. 257, 8363-8367). We have now evaluated the quantitative importance of pathway(s) to cholestanol with 7 alpha-hydroxylated C27 steroids as intermediates. After feeding conventionally fed rabbits or rats or germ-free rats with [7 alpha-3H]cholesterol and [4-14C]cholesterol, tissue cholestanol could be isolated with about a 20% lower 3H/14C ratio than present in cholesterol. We conclude that there is a pathway to cholestanol involving 7 alpha-hydroxylated intermediates. Intestinal microorganisms are not essential for this pathway, which accounts for at most 20% of the cholestanol formed in these species. In bile fistula rats, there was also a significant conversion of intraperitoneally injected [7 beta-3H]7 alpha-hydroxycholesterol and [4-14C]7 alpha-hydroxy-4-cholesten-3-one into cholestanol. The enzymes involved in the 7 alpha-hydroxylation/dehydroxylation pathway for the biosynthesis of cholestanol are probably located in the liver. Both 7 alpha-hydroxycholesterol and 7 alpha-hydroxy-4-cholesten-3-one may be intermediates.     

Simultaneous measurement of cholesterol 7 alpha-hydroxylase activity by reverse-phase high-performance liquid chromatography using both endogenous and exogenous [4-14C]cholesterol as substrate

The HPLC-spectrophotometric method (T. Ogishima and K. Okuda (1986) Anal. Biochem. 158, 228-232) for measuring cholesterol 7 alpha-hydroxylase activity was modified by using a C-18 reverse-phase column to separate 7 alpha-hydroxy-4-cholesten-3-one and 4-cholesten-3-one and by adding 7 beta-hydroxycholesterol to each reaction mixture as an internal recovery standard. With this method, we were able to simultaneously measure cholesterol 7 alpha-hydroxylase activity using endogenous cholesterol and exogenous [4-14C]cholesterol as substrate. Rat liver cytosol differentially stimulated (286%) the 7 alpha-hydroxylation of exogenous [4-14C]-cholesterol. In contrast, total cholesterol 7 alpha-hydroxylase activity was stimulated only 35% by cytosol. This method should prove useful for studying mechanisms of cholesterol delivery to cholesterol 7 alpha-hydroxylase.     

Conversion of 7alpha-hydroxycholesterol and 7alpha-hydroxy-beta-sitosterol to 3alpha, 7alpha-dihydroxy- and 3alpha, 7alpha, 12alpha-trihydroxy-5beta-steroids in vitro.

The metabolism of 7alpha-hydroxycholesterol and 7alpha-hydroxy-beta-sitosterol (24alpha-ethyl-5-cholestene-3beta,7alpha-diol) has been compared in rat liver subcellular fractions. 7alpha-Hydroxy-beta-sitosterol was shown to be metabolized in the same manner as 7alpha-hydroxycholesterol. Thus, the following C29 metabolites have been identified: 24alpha-ethyl-7alpha-hydroxy-4-cholesten-3-one, 24alpha-ethyl-7alpha,12alpha-dihydroxy-4-cholesten-3-one, 24alpha-ethyl-7alpha-hydroxy-5beta-cholestan-3-one, 24alpha-ethyl-5beta-cholestane-3alpha,7alpha-diol, 24alpha-ethyl-7alpha,12alpha-dihydrozy-5beta-cholestan-3-one, and 24alpha-ethyl-5beta-cholestane-3alha,7alpha,12alpha-triol. The C29 compounds were generally less efficient substrates. The most pronounced difference was noted for the delta4-3-oxosteroid 5beta-reductase. Thus, 7alpha-hydroxy-4-cholesten-3-one was three to four times as efficiently reduced as the C29 analog. The oxidation of the 3beta,7alpha-dihydroxy-delta5-steroid to the 7alpha-hydroxy-delta4-3-oxosteroid, the 12alpha-hydroxylation of the 7alpha-hydroxy-delta4-3-oxosteroid, and the reduction of the 7alpha-hydroxy-5beta-3-oxosteroid to the 3alpha,7alpha-dihydroxy-5beta-steroid occurred in up to two times better yields for the C27 steroids.     

Bile acid biosynthesis: the metabolism of 7 alpha-hydroxy-4-cholesten-3-one in the bile fistula rat.

The two primary bile acids, cholic acid (3α,7α,12α-tri-hydroxy-5β-cholan-24-oic acid) and chenodeoxycholic acid (3α,7α-dihydroxy-5β-cholan-24-oic acid), are initially synthesized by way of identical precursors, and the point of divergence of this pathway is thought to occur at the intermediate 7α-hydroxy-4-cholesten-3-one. In order to test this hypothesis, bile fistula rats received simultaneous intra-venous infusions of ³H-7α-hydroxy-4-cholesten-3-one and ¹⁴C-cholesterol (5-cholesten-3β-ol). Assays of equal specific activities of the two bile acids from an infusion of ¹⁴C-cholesterol demonstrated the achievement of a steady state, and assays of equal specific activities from an infusion of ³H-7α-hydroxy-4-cholesten-3-one would-validate the above postulate. However, the infusion of ³H-7α-hydroxy-4-cholesten-3-one resulted in unequal specific activities in the bile acids of the rats investigated, with cholic acid always of a lower value. These results suggest that either 7α-hydroxy-4-cholesten-3-one is not the last common intermediate in the production of cholic acid and chenodeoxycholic acid, or that the infused bile acid intermediate was not metabolized in a fashion similar to that formed in the liver from cholesterol.     

Monitoring hepatic cholesterol 7alpha-hydroxylase activity by assay of the stable bile acid intermediate 7alpha-hydroxy-4-cholesten-3-one in peripheral blood

We describe an accurate method for monitoring the enzymatic activity of hepatic cholesterol 7alpha-hydroxylase (C7alphaOH; CYP7A1), the rate-limiting and major regulatory enzyme in the synthesis of bile acids. Assay of 7alpha-hydroxy-4-cholesten-3-one (C4), an intermediate in bile acid synthesis, revealed that the level of C4 in peripheral blood serum or plasma showed a strong correlation to the enzymatic activity of hepatic C7alphaOH, both at steady-state conditions (r = 0.929) as well as during the rapid changes that occur during the diurnal phases. This assay should be of value in clarifying the regulation of bile acid synthesis in vivo in laboratory animals and humans since it allows for the monitoring of hepatic C7alphaOH activity using peripheral blood samples.     

Metabolism of potential precursors of chenodeoxycholic acid in cerebrotendinous xanthomatosis (CTX).

In patients with cerebrotendinous xanthomatosis (CTX), diminished cholic acid production is associated with incomplete oxidation of the cholesterol side chain and the excretion of C(25)-hydroxy bile alcohols. The aims of this investigation were 1) to provide quantitative information on the pool size and production rate of chenodeoxycholic acid by the isotope dilution technique; and 2) to investigate the possible existence of a block in chenodeoxycholic acid synthesis and explain the absence of chenodeoxycholic acid precursors in CTX. After the injection of [24-(14)C]chenodeoxycholic acid, measurements of chenodeoxycholic acid pool size and production rate in a CTX subject were, respectively, 1/20 and 1/6 as great as controls. Further, three potential precursors of chenodeoxycholic acid, namely [G-(3)H]7alpha-hydroxy-4-cholesten-3-one, [G-(3)H]5beta-cholestane-3alpha,7alpha,25-triol, and [G-(3)H]5beta-cholestane-3alpha,7alpha,26-triol, were administered to the CTX and control subjects and the specific activity curves of [G-(3)H]cholic acid and [G-(3)H]chenodeoxycholic acid were constructed and compared. In the control subjects, the two bile acids decayed exponentially, but in the CTX patient maximum specific activities were abnormally delayed, indicating the hindered transformation of precursor into bile acid. These results show that chenodeoxycholic acid synthesis is small in CTX and that the conversion of 7alpha-hydroxy-4-cholesten-3-one, 5beta-cholestane-3alpha,7alpha,25-triol, and 5beta-cholestane-3alpha,7alpha,26-triol to both chenodeoxycholic acid and cholic acid were similarly impaired.     

Ketonic bile acids in urine of infants during the neonatal period

Ketonic bile acids have been found to be quantitatively important in urine of healthy infants during the neonatal period. In order to determine their structures, the bile acids in urine from 11 healthy infants were analyzed by gas-liquid chromatography-mass spectrometry (GLC-MS) and three samples with particularly high levels of ketonic bile acids were selected for detailed studies by ion exchange chromatography, fast atom bombardment mass spectrometry, microchemical reactions, and GLC-MS. The major ketonic bile acid was identified as 7 alpha, 12 alpha-dihydroxy-3-oxo-5 beta-chol-1-enoic acid, not previously described as a naturally occurring bile acid. The positional isomer 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholenoic acid, recently described as a major urinary bile acid in infants with severe liver diseases, was also excreted by most infants. Three acids related to cholic acid were identified: 7 alpha, 12 alpha-dihydroxy-3-oxo-, 3 alpha, 12 alpha-dihydroxy-7-oxo-, and 3 alpha, 7 alpha-dihydroxy-12-oxo-5 beta-cholanoic acids. Five bile acids having one oxo and three hydroxy groups were also present. Based on mass spectra and biological considerations two of these were tentatively given the structures 1 beta, 7 alpha, 12 alpha-trihydroxy-3-oxo- and 1 beta, 3 alpha, 12 alpha-trihydroxy-7-oxo-5 beta-cholanoic acids. Some of the others had a hydroxy group at C-4 or C-2. The levels of ketonic bile acids were higher on the third than on the first day of life, and lower after 1 month. The formation and excretion especially of 3-oxo bile acids is proposed to result from changes of the redox state in the liver in connection with birth.