Bile Acid Synthesis In Man Research Articles

Highly sensitive quantification of 7α-hydroxy-4-cholesten-3-one in human serum by LC-ESI-MS/MS

We describe a highly sensitive and specific method for the quantification of serum 7alpha-hydroxy-4-cholesten-3-one (C4), which has been used as a biomarker for bile acid biosynthesis. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). C4 was extracted from human serum (2-50 mul) by a salting-out procedure, derivatized into the picolinoyl ester (C4-7alpha-picolinate), and then purified using a disposable C(18) cartridge. The resulting picolinoyl ester derivative of C4 was quantified by LC-MS/MS using the electrospray ionization mode. The detection limit of the C4 picolinoyl ester was found to be 100 fg (signal-to-noise ratio = 10), which was approximately 1,000 times more sensitive than the detection limit of C4 with a conventional HPLC-ultraviolet method. The relative standard deviations between sample preparations and between measurements by our method were calculated to be 5.7% and 3.9%, respectively, by one-way layout analysis. The recovery experiments were performed using serum spiked with 20.0-60.0 ng/ml C4 and were validated by a polynomial equation. The results showed that the estimated concentration with 95% confidence limit was 23.1 +/- 2.8 ng/ml, which coincided completely with the observed X(0) +/- SD = 23.3 +/- 1.0 ng/ml with a mean recovery of 93.4%. This method provides highly reliable and reproducible results for the quantification of C4, especially in small volumes of blood samples.

Circulating intestinal fibroblast growth factor 19 has a pronounced diurnal variation and modulates hepatic bile acid synthesis in man

Bile acids (BAs) traversing the enterohepatic circulation exert several important metabolic effects. Their hepatic synthesis, controlled by the enzyme cholesterol 7alpha-hydroxylase (CYP7A1), has a unique diurnal variation in man. Here we provide evidence that the transintestinal flux of BAs regulates serum levels of intestinal fibroblast growth factor 19 (FGF19) that in turn modulate BA production in human liver. Basal FGF19 levels varied by 10-fold in normal subjects, and were reduced following treatment with a BA-binding resin and increased upon feeding the BA chenodeoxycholic acid. Serum FGF19 levels exhibited a pronounced diurnal rhythm with peaks occurring 90-120 min after the postprandial rise in serum BAs. The FGF19 peaks in turn preceded the declining phase of BA synthesis. The diurnal rhythm of serum FGF19 was abolished upon fasting. We conclude that, in humans, circulating FGF19 has a diurnal rhythm controlled by the transintestinal BA flux, and that FGF19 modulates hepatic BA synthesis. Through its systemic effects, circulating FGF19 may also mediate other known BA-dependent effects on lipid and carbohydrate metabolism.

Mutations in the Gene Encoding Peroxisomal Sterol Carrier Protein X (SCPx) Cause Leukencephalopathy with Dystonia and Motor Neuropathy

In this report, we describe the first known patient with a deficiency of sterol carrier protein X (SCPx), a peroxisomal enzyme with thiolase activity, which is required for the breakdown of branched-chain fatty acids. The patient presented with torticollis and dystonic head tremor as well as slight cerebellar signs with intention tremor, nystagmus, hyposmia, and azoospermia. Magnetic resonance imaging showed leukencephalopathy and involvement of the thalamus and pons. Metabolite analyses of plasma revealed an accumulation of the branched-chain fatty acid pristanic acid, and abnormal bile alcohol glucuronides were excreted in urine. In cultured skin fibroblasts, the thiolytic activity of SCPx was deficient, and no SCPx protein could be detected by western blotting. Mutation analysis revealed a homozygous 1-nucleotide insertion, 545_546insA, leading to a frameshift and premature stop codon (I184fsX7).

Bile Acid Synthesis in Humans Has a Rapid Diurnal Variation That Is Asynchronous With Cholesterol Synthesis

The conversion of cholesterol to bile acids by the liver is an important regulator of body cholesterol homeostasis. In rodents, both cholesterol and bile acid synthesis have marked diurnal rhythms that peak synchronously at midnight. The aim of this study was to establish whether such diurnal rhythms are also present in healthy humans. Serum levels of the markers 7alpha-hydroxy-4-cholesten-3-one (C4) monitoring bile acid biosynthesis and lathosterol reflecting cholesterol synthesis were determined at 90-minute intervals in 8 human volunteers during standardized dietary conditions. Serum C4 showed 2 distinct peaks (2- to 4-fold above baseline) during a 24-hour period, the first at 1:00 pm and the second at 9:00 pm. During the night, C4 levels declined, and they returned to baseline levels the next morning. In contrast, serum lathosterol levels peaked at night, between midnight and 4:00 am. The diurnal changes of C4 were not synchronous with serum lipid changes or with the postprandial increase in serum bile acids and were maintained in cholecystectomized subjects. Bile acid synthesis in humans has a diurnal rhythm, with 2 peaks during the daytime, that is opposite from the circadian rhythm of cholesterol synthesis. This is completely different from the pattern in rodents and indicates the presence of an important species variation in the regulation of cholesterol homeostasis.

Serum concentrations of 7α-hydroxy-4-cholesten-3-one reflect bile acid synthesis in man: [formula omitted] Department of Medicine II, Klinikum Grosshadern, University of Munich, Munich, Germany

Serum concentrations of 7α-hydroxy-4-cholesten-3-one reflect bile acid synthesis in man: [formula omitted] Department of Medicine II, Klinikum Grosshadern, University of Munich, Munich, Germany

Levels of 7α-hydroxy-4-cholesten-3-one in plasma reflect rates of bile acid synthesis in man

A method for analysis of 7α-hydroxy-4-cholesten-3-one in plasma is described. Following solid-phase extraction/purification the compound is determined by high-performance liquid chromatography using a UV detector. The median concentration in healthy subjects was 12 ng/ml (range 3–40). The levels were lower in diseases associated with a low bile acid production: extrahepatic cholestasis, <1.5 ng/ml (range < 0.9–3); liver cirrhosis <1.5 ng/ml (range < 0.9–38), and higher in diseases associated with a high bile acid production: cholestyramine treatment, 188 ng/ml (range 54–477); ileal resection 397 ng/ml (range 128–750). The levels were essentially normal in patients with colon resection. The results are consistent with a strong positive correlation between the levels of 7α-hydroxy-4-cholesten-3-one in plasma and the rate of bile acid synthesis.

Bile acid synthesis in man. In vivo activity of the 25-hydroxylation pathway.

During biosynthesis of bile acid, carbons 25-26-27 are removed from the cholesterol side-chain. Side-chain oxidation begins either with hydroxylation at the 26-position, in which case the three-carbon fragment is released as propionic acid, or with hydroxylation at the 25-position, in which case the three-carbon fragment is released as acetone. We have previously shown in the rat that the contribution of the 25-hydroxylation pathway can be quantitated in vivo by measuring production of [14C]acetone from [14C]26-cholesterol. In the present study, we adapted this method to human subjects. 4 d after oral administration of 100 microCi of [14C]26-cholesterol and 1 d after beginning a constant infusion of 16.6 mumol/min unlabeled acetone, three men and two women underwent breath collections. Expired acetone was trapped and purified as the 2,4 dinitrophenylhydrazine derivative. 14CO2 was trapped quantitatively using phenethylamine. Specific activity of breath acetone was multiplied by the acetone infusion rate to calculate production of [14C]acetone. [14C]Acetone production averaged 4.9% of total release of 14C from [14C]26-cholesterol, estimated by 14CO2 output. The method was validated by showing that [14C]acetone production from [14C]isopropanol averaged 86.9% of the [14C]-isopropanol infusion rate. We conclude that in man, as in the rat, the 25-hydroxylation pathway accounts for less than 5% of bile acid synthesis.

Deficient cholesterol side chain oxidation in patients without peroxisomes (Zellweger syndrome): evidence for the involvement of peroxisomes in bile acid synthesis in man

The absence of peroxisomes in patients with the cerebro-hepato-renal (Zellweger) syndrome is accompanied by a number of biochemical abnormalities including the accumulation of the bile acid intermediates di- and trihydroxycoprostanoic acid. In this paper we show that there is a marked deficiency in the oxidative side chain cleavage of cholesterol in liver from Zellweger patients. These findings not only provide an explanation for the low levels of the major naturally occurring bile acids, cholic acid and chenodeoxycholic acid and the accumulation of di- and trihydroxycoprostanoic acid in Zellweger patients, but also suggest that peroxisomes are essential in bile acid synthesis in man.

Effect of bile acid structure on the feed-back regulation of bile acid synthesis in man: Studies on cholesterol 7 α-hydroxylation

Effect of bile acid structure on the feed-back regulation of bile acid synthesis in man: Studies on cholesterol 7 α-hydroxylation

Bile acid synthesis in man: assay of hepatic microsomal cholesterol 7 alpha-hydroxylase activity by isotope dilution-mass spectrometry.

The present work describes an accurate assay of the rate-limiting enzyme in bile acid synthesis, the cholesterol 7 alpha-hydroxylase, in human liver. The assay is based on isotope dilution-mass spectrometry, and endogenous microsomal cholesterol is used as the only substrate for the enzyme. Operative liver biopsies were obtained from patients undergoing elective cholecystectomy under highly standardized conditions. In ten gallstone patients, the enzyme activity of the microsomal fraction averaged 9.6 +/- 1.4 (mean +/- SEM) pmol X min-1 X mg protein-1 corresponding to a daily synthesis of about 0.5 mmol of bile acids. Three cholestyramine-treated patients displayed a four-fold higher enzyme activity. No evidence was obtained supporting the concept that the cholesterol 7 alpha-hydroxylase is modulated by phosphorylation-dephosphorylation.

Regulation of bile acid synthesis in man. Presence of a diurnal rhythm.

Regulation of bile acid synthesis in man is incompletely understood, in part because of difficulty in making measurements over short time periods when the enterohepatic circulation is intact. We investigated the possibility of a diurnal rhythm of bile acid synthesis in three human subjects given [26-14C]cholesterol. When this isotope of cholesterol, which is randomly labeled in the 26 and 27 positions, is converted to bile acid, the 14C is released as propionic acid randomly labeled in the 1 and 3 positions. The labeled propionic acid is then oxidized to 14CO2, output of which is a function of bile acid synthesis. However, delays in transit of the 14C through propionic acid and CO2-HCO-3 pools would shift the phase and dampen the amplitude of 14CO2 output relative to an existing diurnal rhythm of bile acid synthesis. Therefore, using constant infusion methods, we determined the turnover constants for conversion to 14CO2 of [1-14C]propionic acid and [3-14C]propionic acid to be 0.36-0.59 h-1 and 0.14-0.16 h-1, respectively. Using these constants and modeling the diurnal rhythm as a cosine function, we determined that amplitude of 14CO2 output from [26-14C]cholesterol was reduced 35% and acrophase was delayed 2.4-3.0 h relative to the diurnal rhythm of bile acid synthesis. None of the diurnal rhythm in 14CO2 output from [26-14C]cholesterol resulted from diurnal variation in propionic acid or CO2-HCO-3 metabolism since constant infusion of [1-14C]propionic acid and [3-14C]propionic acid for 30 h revealed no diurnal variation in output of 14CO2. These studies demonstrate for the first time that humans with an intact enterohepatic circulation have a diurnal rhythm of bile acid synthesis with an amplitude of +/- 35-55% around mean synthesis, and an acrophase at about 9 a.m.

Bile acid synthesis in man III. Identification of 26-hydroxycholesterol in serum in zellweger syndrome

Bile acid synthesis in man III. Identification of 26-hydroxycholesterol in serum in zellweger syndrome

Evidence for a Common Hepatic Cholesterol Precursor Site for Cholic and Chenodeoxycholic Acid Synthesis in Man

SummaryBile fistula patients were administered [14C]cholesterol and labeled mevalonic acid by constant infusion (for 5 hr) or by intravenous pulse. Bile was collected at frequent time intervals. The specific activities of newly synthesized cholic and chenodeoxycholic acids were virtually identical at all time periods (up to 24 hr). These observations provide evidence for the presence of a common cholesterol substrate pool for primary bile acid synthesis in man. Both cholic and chenodeoxycholic acids appear to be synthesized via the 7α-hydroxy-cholesterol pathway.

Bile acid synthesis in man. II. Determination of 7 α-hydroxycholesterol, (22R)-22-hydroxycholesterol, and 26-hydroxycholesterol in human meconium

7 alpha-Hydroxycholesterol, (22R)-22-hydroxycholesterol and 26-hydroxycholesterol have been quantitated in human meconium. The method used tetrahydrofuran for extraction and solvolysis of the sulfate esters, liquid partition chromatography for the separation of the hydroxysterols, gas-liquid chromatography for quantitation, gas-liquid chromatography-mass spectrometry for identification, and tritiated and 14C-labeled tracers for overall recovery standards. (22R)-22-Hydroxycholesterol and 26-hydroxycholesterol were present almost entirely, ( greater than 93%) in the sulfate fraction at concentrations of 3.8-6.4 and 0.4-0.8 mg per 100 g meconium, respectively. Since free tritiated (22R)-22-hydroxycholesterol was used as the tracer to assess recovery of this hydroxysterol, the concentrations found for this compound may be minimal. Tritiated 26-hydroxycholesterol 3,26-disulfate was used as tracer to determine the levels of this compound, and the solvolysis procedure was optimized for recovery of 26-hydroxycholesterol and least decomposition of 7 alpha-hydroxycholesterol. No significant amounts of 7 alpha-hydroxycholesterol were found based on the tracer-free hydroxysterol as recovery standard.

Rapid feedback inhibition of endogenous cholic and chenodeoxycholic acid synthesis by exogenous chenodeoxycholic acid in man

Chenodeoxycholic acid (300 mg + 14C) was administered orally to a bile fistula patient receiving a constant infusion of { 3H}mevalonic acid. Suppression of endogenous cholic and chenodeoxycholic acid synthesis occurred within 2 to 4 hours and continued for the next 10 hours; synthesis returned to the baseline level after 18 hours. Incorporation of { 3H}mevalonic acid into both bile acids was also greatly reduced during the first several hours after chenodeoxycholic acid, but almost recovered by 5 hours. The data suggest that multiple feedback sites are involved in the regulation of bile acid synthesis in man.

Bile acid synthesis in man: metabolism of 7 -hydroxycholesterol- 14 C and 26-hydroxycholesterol- 3 H.

The pathways of bile acid synthesis in man were evaluated by studying the metabolism of 7alpha-hydroxycholesterol-4-(14)C and 26-hydroxycholesterol-16, 22-(3)H administered parenterally to individuals requiring external biliary drainage. Techniques for the identification of metabolites were thin-layer chromatography, column chromatography, gas-liquid chromatography with stream splitting, and crystallization to constant specific activity. It was found that both compounds were rapidly metabolized to bile acids and excreted in bile. Of the total radioactivity recovered in bile as bile acids, 87% of the 26-hydroxycholesterol-(3)H and 90% of the 7alpha-hydroxycholesterol-(14)C was found to be metabolized to both chenodeoxycholate and cholate. Compared to 7alpha-hydroxycholesterol, a greater proportion of 26-hydroxycholesterol was found to be metabolized to chenodeoxycholate. These findings indicate that both 7alpha-hydroxycholesterol and 26-hydroxycholesterol can be intermediates in the metabolism of cholesterol to bile acids in man. The observation that conversion to cholate takes place less readily after C-26 hydroxylation is consistent with previous findings in other species.