3-7-dihydroxycholestan-26-oic-acid and 3-7-12-trihydroxycholestan-26-oic-acid

A subgroup of peroxisomal disorders, peroxisome biogenesis defects (PBD), can be differentiated by elevated levels of C(27) bile acids in plasma and bile. Patients with peroxisomal disorders, who lack the ability to chain-shorten the C(27) bile acid intermediates into C(24) bile acids, show elevated levels of C(27) bile acids, notably 3 alpha,7 alpha-dihydroxy-5 beta-cholest-26-oic acid and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid. C(27) bile acids are normally estimated against other bile acid standards, by time-consuming gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry methods, in plasma (minimum of 50 microl). In this article we describe the quantitation of unconjugated di- and trihydroxy C(27) bile acids in 5-microl plasma samples and 3-mm blood spots, using deuterium-labeled internal standards. The synthesis of (2)H(3)-labeled di- and trihydroxycoprostanic acids is described. The sample preparation and analysis by electrospray tandem mass spectrometry (ES-MS/MS) takes less than 1 h and features dimethylaminoethyl ester derivatives. The levels of the di- and trihydroxy bile acids are significantly higher in PBD patients than in age-matched control subjects for both plasma and blood spots collected at birth (some stored for up to 18 years). Excellent correlation is observed between the C(26:0)/C(22:0) very long chain fatty acid (VLCFA) ratio and the levels of trihydroxy C(27) bile acids in plasma from PBD patients. The ES-MS/MS method can be used to rapidly screen for PBD patients in plasma samples with elevated C(26:0)/C(22:0) VLCFA ratios and in archived collections of neonatal blood spots. - Johnson, D. W., H. J. ten Brink, R. C. Schuit, and C. Jakobs. Rapid and quantitative analysis of unconjugated C(27) bile acids in plasma and blood samples by tandem mass spectrometry. J. Lipid Res. 2001. 42: 9;-16.

Topics: Age Factors; Bile Acids and Salts; Child; Child, Preschool; Cholestanols; Deuterium; Fatty Acids; Humans; Infant; Infant, Newborn; Matched-Pair Analysis; Peroxisomal Disorders; Spectrometry, Mass, Electrospray Ionization

We identified a new peroxisomal disorder caused by a deficiency of the enzyme alpha-methylacyl-coenzyme A (CoA) racemase. Patients with this disorder show elevated plasma levels of pristanic acid and the bile acid intermediates di- and trihydroxycholestanoic acid (DHCA and THCA), which are all substrates for the peroxisomal beta-oxidation system. alpha-Methylacyl-CoA racemase plays an important role in the beta-oxidation of branched-chain fatty acids and fatty acid derivatives because it catalyzes the conversion of several (2R)-methyl-branched-chain fatty acyl-CoAs to their (2S)-isomers. Only stereoisomers with the 2-methyl group in the (S)-configuration can be degraded via beta-oxidation. In this study we used liquid chromatography/tandem mass spectrometry (LC-MS/MS) to analyze the bile acid intermediates that accumulate in plasma from patients with a deficiency of alpha-methylacyl-CoA racemase and, for comparison, in plasma from patients with Zellweger syndrome and patients with cholestatic liver disease.We found that racemase-deficient patients accumulate exclusively the (R)-isomer of free and taurine-conjugated DHCA and THCA, whereas in plasma of patients with Zellweger syndrome and patients with cholestatic liver disease both isomers were present. On the basis of these results we describe an easy and reliable method for the diagnosis of alpha-methylacyl-CoA racemase-deficient patients by plasma analysis. Our results also show that alpha-methylacyl-CoA racemase plays a unique role in bile acid formation. - Ferdinandusse, S., H. Overmars, S. Denis, H. R. Waterham, R. J. A. Wanders, and P. Vreken. Plasma analysis of di- and trihydroxycholestanoic acid diastereoisomers in peroxisomal alpha-methylacyl-CoA racemase deficiency. J. Lipid Res. 2001. 42: 137;-141.

Topics: Bile Acids and Salts; Child, Preschool; Cholestanols; Cholestasis, Intrahepatic; Diagnosis, Differential; Female; Humans; Infant; Male; Oxidation-Reduction; Peroxisomal Disorders; Peroxisomes; Racemases and Epimerases; Spectrometry, Mass, Electrospray Ionization; Stereoisomerism; Zellweger Syndrome

A new method for the preparation of 5beta-cholestan-26-oic acids 7 and their analogs is described. The key steps in the synthesis are: iodination of bis- and tris-formyloxy-5beta-cholan-24-ols 3; nucleophilic substitution of iodides 4 with diethyl sodiomalonate; complete alkaline hydrolysis of esters 5; and subsequent decarboxylation of geminal diacids 6 in DMSO.

Topics: Cholestanols

The biosynthetic intermediates of bile acid, 3 alpha, 7 alpha, 12 alpha-trihydroxy- and 3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acids, were synthesized by means of the thermal elimination of beta-ketosulfoxides. The alpha, beta-unsaturated ketones as key compounds of the synthesis, 3 alpha, 7 alpha, 12 alpha-trihydroxy- and 3 alpha, 7 alpha-dihydroxy-5 beta-cholest-25-en-24-ones, were effectively derived from the beta-ketosulfoxides prepared from methyl cholate or chenodeoxycholate by reaction with methylsulfinylcarbanion. These unsaturated ketones were converted into 3 alpha, 7 alpha, 12 alpha, 26-tetrahydroxy- and 3 alpha, 7 alpha, 26-trihydroxy-5 beta-cholestanes by reductive deoxygenation and hydroboration, of which stereoisomers were chromatographically separated into 25S- and 25R- isomers. The oxidation of each of the above isomeric alcohols after the protection of the hydroxyl groups on the steroidal ring and the following hydrolysis gave the title 26-carboxylic acids.

Topics: Chenodeoxycholic Acid; Cholates; Cholestanols; Cholic Acids; Indicators and Reagents; Ketones; Magnetic Resonance Spectroscopy; Sulfoxides

Topics: Cholestanols; Fatty Acids; Humans; Oxidoreductases; Phytanic Acid

We have examined the ability of HepG2 human hepatoblastoma cells and 7800 C1 Morris rat hepatoma cells to convert 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) and 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA) to cholic acid and chenodeoxycholic acid, respectively. Cell extracts from both these cell lines could neither form cholic acid from THCA nor from the activated form, THCA-CoA. This suggests that both cell lines are defective in two enzyme activities involved in the pathway, the microsomal THCA-CoA ligase and the peroxisomal THCA-CoA oxidase. Furthermore, we show that the subsequent enzymes are active in the conversion to bile acids, because the product of the THCA-CoA oxidase, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholest-24-enoyl-coenzyme A (delta 24-THCA-CoA) or delta 24-THCA in the presence of THCA-CoA ligase, are converted to cholic acid by both cell lines. HepG2 cells were able to slowly form chenodeoxycholic acid and cholic acid from 5 beta-cholestane-3 alpha, 7 alpha-diol and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, respectively, in 24- and 96-h incubations. The rate of cholic acid formation was lower than the rate for chenodeoxycholic acid and there was a clear accumulation of THCA. 7800 C1 Morris cells had no ability to form cholic acid or chenodeoxycholic acid after 96 h incubation. We conclude that these two cell lines have defects in two enzyme activities involved in the peroxisomal oxidation in bile acid formation, the microsomal THCA-CoA ligase and the peroxisomal THCA-CoA oxidase.

Topics: Animals; Bile Acids and Salts; Chenodeoxycholic Acid; Cholestanols; Cholesterol 7-alpha-Hydroxylase; Cholic Acid; Cholic Acids; Clofibric Acid; Dexamethasone; Fatty Acids; Hepatoblastoma; Humans; Liver Neoplasms; Liver Neoplasms, Experimental; Male; Oxidation-Reduction; Rats; Rats, Wistar; Retinoids; Tumor Cells, Cultured

Synthesis of 25R- and 25S-diastereoisomers of 3 alpha,7 alpha-dihydroxy-5 beta-cholestan-26-oic acid from 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid is described. The 25S-diastereoisomer of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan- 26-oic acid was obtained by vigorous hydrolysis of the bile of Alligator mississippiensis followed by repeated crystallization of the hydrolysate, and the 25R-diastereoisomer was isolated by hydrolysis of the bile salts in bile of A mississippiensis with rat feces. Acetylation of the 25R- or 25S-diastereoisomer of methyl 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid under controlled conditions yielded the corresponding 3 alpha,7 alpha-diacetate in approximately 70% yield. The diacetate was quantitatively oxidized to methyl 3 alpha,7 alpha-diacetoxy-12-oxo-5 beta-cholestan-26-oate, which was converted into the 12-tosylhydrazone in approximately 58% yield. Reduction of the tosylhydrazone with sodium borohydride in acetic acid yielded the 25R- or the 25S-diastereoisomer of 3 alpha,7 alpha-dihydroxy-5 beta-cholestan-26-oic acid as the major product. Purification via column chromatography yielded the pure diastereoisomers in approximately 25% overall yield. The two diastereoisomers were resolved on thin-layer chromatography and high-performance liquid chromatography. When the bile of A mississippiensis was hydrolyzed with rat fecal bacteria, the 3 alpha,7 alpha-dihydroxy-5 beta-cholestan-26-oic acid isolated via chromatographic purification was shown to be the 25R-diastereoisomer.

Topics: Acetylation; Alligators and Crocodiles; Animals; Bile; Cholestanols; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Gas Chromatography-Mass Spectrometry; Magnetic Resonance Spectroscopy; Stereoisomerism

Topics: Amniotic Fluid; Bile Acids and Salts; Chenodeoxycholic Acid; Cholestanols; Cholic Acids; Female; Fetus; Humans; Metabolism, Inborn Errors; Microbodies; Pregnancy; Prenatal Diagnosis

Two patients with a suspected peroxisomal disorder on the basis of neurological, craniofacial, hepatological and other abnormalities were studied. The phenotype of both girls was remarkably similar from birth until age 1.5 yr. Detailed studies in plasma revealed normal plasma very-long-chain fatty acids but the presence of di- and trihydroxycholestanoic acids and the C29-dicarboxylic bile acid, all known to occur in plasma from Zellweger patients. These results suggest an isolated defect in the peroxisomal beta-oxidation of the side chains of the cholestanoic acids. Activation of trihydroxycholestanoic acid and beta-oxidation of trihydroxycholestanoyl-CoA, measured in a liver biopsy, were normal, however, as was the peroxisomal beta-oxidation of palmitate. Although the molecular defect remains unknown, the results stress the importance of performing multiple analyses in any patient suspected to suffer from a peroxisomal disorder and indicate that screening for peroxisomal disorders based upon analysis of only plasma very long chain fatty acids with or without analysis of erythrocyte plasmalogen levels, may be inadequate.

Topics: Abnormalities, Multiple; Acyl Coenzyme A; Bile Acids and Salts; Cells, Cultured; Cholestanols; Coenzyme A Ligases; Dicarboxylic Acids; Diseases in Twins; Fatty Acids, Nonesterified; Female; Fibroblasts; Humans; Infant; Liver; Microbodies; Palmitoyl Coenzyme A; Phenotype; Repressor Proteins; Saccharomyces cerevisiae Proteins; Skin; Twins, Dizygotic; Zellweger Syndrome

The high-performance liquid chromatographic separation of the 25R and 25S diastereoisomers of the bile alcohols 5 beta-cholestane-3 alpha,7 alpha,26-triol and 5 beta-cholestane-3 alpha,7 alpha, 12 alpha, 26-tetrol and the bile acids, 3 alpha,7 alpha-dihydroxy-5 beta-cholestane-26-oic acid and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestane-26-oic acid is described. A Radial-Pak microBondapak C18 reversed-phase cartridge was used for the separations and elutions were carried out with acetonitrile-water-methanol-acetic acid mixtures. All eight diastereoisomeric compounds showed baseline separation when up to 200 micrograms of the isomeric mixtures were injected into the column and the method can be used for isolation of pure diastereoisomers of these bile acids and bile alcohols.

Topics: Alligators and Crocodiles; Animals; Bile; Bile Acids and Salts; Cholestanols; Chromatography, High Pressure Liquid; Stereoisomerism

A method for the determination of 3 alpha,7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA) and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) in human urine by gas chromatography (GC) in combination with negative ion chemical ionization (NICI) mass spectrometry is described. Unconjugated, glycine- and taurine-conjugated DHCA and THCA labelled with 18O and 2H were used as internal standards. 5 beta-Cholestanoic acids in urine were extracted with a Sep-Pak C18 cartridge, separated into the unconjugated, glycine- and taurine-conjugated fractions by ion-exchange chromatography on piperidinohydroxypropyl Sephadex LH-20 and, following alkaline hydrolysis of conjugated forms, derivatization into the pentafluorobenzyl ester-dimethylethylsilyl ethers. Subsequent resolution of each fraction into DHCA and THCA was attained by GC on a cross-linked 5% phenylmethylsilicone fused-silica capillary column where 5 beta-cholestanoic acids were monitored with a characteristic carboxylate anion [M-181]- in the NICI mode using isobutane as a reagent gas. The method was applied to separation and determination of 5 beta-cholestanoic acids in urine from a patient with Zellweger syndrome and from healthy volunteers.

Topics: Bile Acids and Salts; Cholestanols; Gas Chromatography-Mass Spectrometry; Humans; Hydrolysis; Steroids

We used capillary gas chromatography/mass spectrometry to demonstrate that a cell line derived from a well differentiated human hepatoblastoma, HepG2, synthesized and secreted the following bile acids (ng/10(7) cells/h): chenodeoxycholic acid (131.4), cholic acid (3.3), 3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acid (DHCA; 4.5), and 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid (THCA; 32.0). Deuterium from [7 beta-2H]7 alpha-hydroxycholesterol, which was added to the media, was incorporated into newly synthesized chenodeoxycholic acid, DHCA, and THCA, but not into cholic acid. Since THCA is a known precursor of cholic acid, these data suggest that HepG2 is specifically deficient in the side chain cleavage that transforms THCA into cholic acid. Greater than 90% of the bile acids synthesized and secreted by HepG2 were unconjugated. Conjugation could not be stimulated by the addition of glycine or taurine to the media. Approximately 30% of newly synthesized DHCA and THCA were sulfated. Chenodeoxycholic acid and cholic acid were not appreciably sulfated. In summary, cultured HepG2 cells synthesize bile acid, but in a pattern distinct from that of adult human liver. This cell line may be a model for studying pathways of human bile acid synthesis, conjugation, and sulfation.

Topics: Bile Acids and Salts; Carcinoma, Hepatocellular; Cell Line; Chenodeoxycholic Acid; Cholestanols; Cholic Acid; Cholic Acids; Gas Chromatography-Mass Spectrometry; Humans; Liver Neoplasms

The oxidation of the side chain of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA) into chenodeoxycholic acid has been studied in subcellular fractions of rat liver. The product was separated from the substrate by high pressure liquid chromatography and identified by gas-liquid chromatography-mass spectrometry. The highest specific rate of conversion was found in the heavy (M) and the light (L) mitochondrial fractions with the highest enrichment in the L fraction. Washing the M fraction reduced the side chain cleavage activity by 90%. The peroxisomal marker enzyme urate oxidase was reduced to the same extent. The activity found in the M fraction may thus be due to peroxisomal contamination. After centrifugation of the L fraction on a Nycodenz density gradient, the highest specific activity for side chain cleavage of DHCA (31 nmol X mg-1 X h-1) was found in the fraction with the highest peroxisomal marker enzyme activity. This fraction also catalyzed conversion of 3 alpha,7 alpha,12 alpha-5 beta-cholestanoic acid (THCA) into cholic acid at the highest rate (32 nmol X mg-1 X h-1). The peroxisomal oxidation of DHCA into chenodeoxycholic acid required the presence of ATP, CoA, Mg2+, and NAD in the incubation medium. The reaction was not inhibited by KCN. It is concluded that rat liver peroxisomes contain enzymes able to catalyze the cleavage of the side chain of both DHCA and THCA. The enzymes involved are similar to, but not necessarily identical to, those involved in the peroxisomal beta-oxidation of fatty acids.

Topics: Animals; Bile Acids and Salts; Chenodeoxycholic Acid; Cholestanols; Cholic Acid; Cholic Acids; In Vitro Techniques; Liver; Male; Microbodies; Oxidation-Reduction; Rats; Rats, Inbred Strains; Subcellular Fractions

3 alpha,7 alpha-dihydroxy-5 beta-cholestan-26-oic acid (DHCA) and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid (THCA) are metabolized into chenodeoxycholic acid and cholic acid, respectively, through oxidation and cleavage of the terminal three carbons of the side chain. The present study was designed to determine if the same or different side chain oxidation systems are used by these compounds in the bile fistula hamster model. Although a single injection of [3H]THCA is nearly completely metabolized into cholic acid, only about 50% is converted into cholic acid when THCA is infused at a rate of 0.083 mumol/min. The remainder is excreted in the bile unchanged indicating saturation of the side chain oxidation system. Fifty-nine +/- 1.1% (+/- 1SEM) of a single injection of [3H]DHCA is metabolized into chenodeoxycholic acid in bile fistula hamsters infused with either saline or cholic acid at a rate of 0.083 mumol/min. The remainder was excreted as several other metabolic products including cholic acid. However, when [3H]DHCA was administered during an 0.083 mumol/min infusion of THCA, only 39.0 +/- 4.5% of the radioactivity in bile was identified as chenodeoxycholic acid. Thus, this study indicates that DHCA and THCA share at least one of the enzymes involved in side chain oxidation.

Topics: Animals; Bile; Binding, Competitive; Chenodeoxycholic Acid; Cholestanols; Cholic Acids; Cricetinae; Isotope Labeling; Male; Mesocricetus; Tritium