Bile acid profiles in bile, urine, and feces of a patient with cerebrotendinous xanthomatosis

Bile acid profiles of bile, urine, and feces obtained from a patient with cerebrotendinous xanthomatosis on the same day have been analyzed by gas-liquid chromatography-mass spectrometry after fractionation into groups by mode of conjugation by an ion-exchange chromatography. The predominant biliary bile acid was cholic acid conjugated with glycine and taurine. Lesser amounts of the amino acid conjugates of chenodeoxycholic acid, ursodeoxycholic acid, 7-ketodeoxycholic acid, allocholic acid, and deoxycholic acid, and of unconjugated norcholic acid and allonorcholic acid were also present in the bile. The major fecal bile acid was 7-epicholic acid. Relatively large amounts of bile acids were excreted in the urine. Unconjugated 7-epicholic acid, norcholic acid, allonorcholic acid, and cholic acid predominated. The bile acid profiles of the patient were different from those of normal subjects and should be useful for the diagnosis.     

Mutations in the bile acid biosynthetic enzyme sterol 27-hydroxylase underlie cerebrotendinous xanthomatosis

The sterol storage disorder cerebrotendinous xanthomatosis (CTX) is characterized by abnormal deposition of cholesterol and cholestanol in multiple tissues. Deposition in the central nervous system leads to neurological dysfunction marked by dementia, spinal cord paresis, and cerebellar ataxia. Deposition in other tissues causes tendon xanthomas, premature atherosclerosis, and cataracts. In two unrelated patients with CTX, we have identified different point mutations in the gene (CYP27) encoding sterol 27-hydroxylase, a key enzyme in the bile acid biosynthesis pathway. Transfection of mutant cDNAs into cultured cells results in the synthesis of immunoreactive sterol 27-hydroxylase protein with greatly diminished enzyme activity. We have localized the CYP27 gene to the q33-qter interval of human chromosome 2, and to mouse chromosome 1, in agreement with the autosomal recessive inheritance pattern of CTX. These findings underscore the essential role played by sterols in the central nervous system and suggest that mutations in other sterol metabolizing enzymes may contribute to diseases with neurological manifestations.     

Increased plasma bile alcohol glucuronides in patients with cerebrotendinous xanthomatosis: effect of chenodeoxycholic acid

Large quantities of C27 bile alcohols hydroxylated at C-25 are excreted in the bile and urine of patients with cerebrotendinous xanthomatosis, a lipid storage disease that results from defective bile acid synthesis. The presence of both biliary and urinary bile alcohols reflects impaired bile acid synthesis. After treatment of samples with beta-glucuronidase, plasma bile alcohols were quantitated by gas-liquid chromatography-mass spectrometry. 5 beta-Cholestane-3 alpha,7 alpha,12 alpha,25-tetrol (334 micrograms/dl) was found to be the major bile alcohol, followed by 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23R,25-pentol (65 micrograms/dl), and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24(R and S),25-pentols (62.5 micrograms/dl and 64.5 micrograms/dl, respectively) in the plasma of these patients. When compared to biliary and urinary bile alcohol excretions, the plasma pattern resembled bile where 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol glucuronide predominated. In contrast, urinary bile alcohols were composed chiefly of 5 beta-cholestanepentol glucuronides with only small amounts of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol glucuronide. Treatment with chenodeoxycholic acid, which suppresses abnormal bile acid synthesis in these patients, reduced plasma bile alcohol concentrations dramatically. These results show that large quantities of bile alcohol glucuronides, particularly 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrolglucuronide, circulate in plasma of patients with cerebrotendinous xanthomatosis. The plasma bile alcohols closely resemble biliary bile alcohols which indicates their hepatic origin. The large quantities of polyhydroxylated bile alcohols in the urine may suggest their formation, at least in part, from 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol by renal hydroxylating mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

A newborn screening method for cerebrotendinous xanthomatosis using bile alcohol glucuronides and metabolite ratios

Cerebrotendinous xanthomatosis (CTX) is a treatable neurodegenerative metabolic disorder of bile acid synthesis in which symptoms can be prevented if treatment with chenodeoxycholic acid supplementation is initiated early in life, making CTX an excellent candidate for newborn screening. We developed a new dried blood spot (DBS) screening assay for this disorder on the basis of different ratios between the accumulating cholestanetetrol glucuronide (tetrol) and specific bile acids/bile acid intermediates, without the need for derivatization. A quarter-inch DBS punch was extracted with methanol, internal standards were added, and after concentration the extract was injected into the tandem mass spectrometer using a 2 min flow injection analysis for which specific transitions were measured for cholestanetetrol glucuronide, taurochenodeoxycholic acid (t-CDCA), and taurotrihydroxycholestanoic acid (t-THCA). A proof-of-principle experiment was performed using 217 Guthrie cards from healthy term/preterm newborns, CTX patients, and Zellweger patients. Using two calculated biomarkers, tetrol:t-CDCA and t-THCA:tetrol, this straightforward method achieved an excellent separation between DBSs of CTX patients and those of controls, Zellweger patients, and newborns with cholestasis. The results of this small pilot study indicate that the tetrol:t-CDCA ratio is an excellent derived biomarker for CTX that has the potential to be used in neonatal screening programs.     

Bile alcohol profiles in bile, urine, and feces of a patient with cerebrotendinous xanthomatosis

Bile alcohols in bile, urine, and feces of a patient with cerebrotendinous xanthomatosis have been analyzed by a combination of capillary gas-liquid chromatography and mass spectrometry after fractionation into groups according to mode of conjugation. The presence of at least 18 bile alcohols, which were excreted mainly as glucurono-conjugates in bile and urine, and as unconjugated forms in feces, was demonstrated. The following bile alcohols were identified with certainty by direct comparison with reference compounds: 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol; (23R)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,23-tetrol; 5 alpha- and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24-tetrols; 5 alpha- and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrols; 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol; (22R)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,22,25-pentol; (23R)- and (23S)-5 beta-cholestane-3 alpha,7 alpha, 12 alpha,23,25-pentols; 3 alpha,12 alpha,25-trihydroxy-5 beta-cholestane-7-one; (24R)- and (24S)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentols; 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol. Although the bile alcohol profile in urine was quite different from those in bile and feces, the determination of urinary bile alcohols as well as of biliary and fecal bile alcohols could be used for diagnosis of cerebrotendinous xanthomatosis.     

Abnormal urinary bile acids in a patient suffering from cerebrotendinous xanthomatosis during oral administration of ursodeoxycholic acid

The urinary bile acid profile, obtained by capillary gas chromatography, of a patient suffering from cerebrotendinous xanthomatosis and treated with ursodeoxycholic acid demonstrated, besides the occurrence of 23-norcholic acid and (23R)-hydroxycholic acid (as a consequence of this disease), six additional unknown bile acids and three known bile acids, viz. ursodeoxycholic acid, hyocholic acid and omega-muricholic acid. The structure of two of the unknown bile acids were elucidated and proven by organic syntheses. These were 23-norursodeoxycholic acid and 3 beta-ursodeoxycholic acid. The structures of three bile acids were tentatively elucidated as being 1 beta-hydroxyursodeoxycholic acid, 21-hydroxyursodeoxycholic acid and 22-hydroxyursodeoxycholic acid, and the possibility that the structure of the remaining bile acid is that of 5-hydroxyursodeoxycholic acid is discussed. Two of these bile acids (1 beta-hydroxyursodeoxycholic acid and 5-hydroxyursodeoxycholic acid) also occurred in urine of a healthy individual during oral ursodeoxycholic acid treatment, whereas 23-norcholic acid, 23-norursodeoxycholic acid, (23R)-hydroxycholic acid, 21-hydroxyursodeoxycholic acid and 22-hydroxyursodeoxycholic acid were only present in urine of the patient suffering from cerebrotendinous xanthomatosis. The metabolism of ursodeoxycholic acid, both in the normal state and in the cerebrotendinous xanthomatosis, is discussed.     

Effect of chenodeoxycholic acid on biliary and urinary bile acids and bile alcohols in cerebrotendinous xanthomatosis; monitoring by high performance liquid chromatography

Biliary and urinary bile alcohol and bile acid composition has been determined by high performance liquid chromatography in patients with cerebrotendinous xanthomatosis before and after treatment with chenodeoxycholic acid. Most of the bile acids and bile alcohols in the bile and urine were separated in less than 30 min using a radial pack C18 muBondapak 5 micron particle size column with a mobile phase of acetonitrile-water-methanol-acetic acid 70:70:20:1 (v/v/v/v) at a flow rate of 2 ml/min, and a refractive index detector. Before treatment, cholic acid (49%) and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol (27%) were the major biliary bile acid and bile alcohol, respectively, but were not detected in the urine of five patients. 5 beta-Cholestane-pentols were, instead, the major urinary bile alcohols with 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 23 xi, 25-pentol (56%) predominating. Whereas 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 24S,25-pentol was not detected in the bile, it was isolated in the urine of all patients (27%). The only urinary bile acid isolated by high performance liquid chromatography was nor-cholic acid. After 1 month of treatment with chenodeoxycholic acid, 0.75 g/day, chenodeoxycholic acid became the major bile acid in the bile of all patients (71%) along with its metabolite, ursodeoxycholic acid (21%). Cholic acid and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol were drastically reduced and were only 3% each. The excretion of 5 beta-cholestane-pentols in the urine was also drastically reduced from 130 mg/day to 15 mg/day.     

Chenodeoxycholic acid normalizes elevated lipoprotein secretion and catabolism in cerebrotendinous xanthomatosis

Cerebrotendinous xanthomatosis (CTX) is a rare inherited lipid storage disease caused by a defect in bile acid synthesis in which cholesterol and its product cholestanol are deposited in neurological and vascular tissue. Therapy with chenodeoxycholic acid but not with the 7 beta-epimeric ursodeoxycholic acid is usually successful. In an untreated patient, total and low density lipoprotein (LDL) cholesterol were found to be low (134 +/- 11 and 78 +/- 8 mg/dl, respectively). The production rate (PR) and fractional catabolic rate (FCR) of very low density (VLDL) apolipoprotein B (apoB) were, however, both markedly increased (34.7 mg/kg per day and 13.7 pools/day, respectively vs. 15.1 +/- 5.0 mg/kg per day and 6.2 +/- 3.8 pools/day in controls) while the PR and FCR of LDL apoB were moderately elevated (16.3 mg/kg per day and 0.65 pools/day, respectively vs. 12.9 +/- 1.2 mg/kg per day and 0.52 +/- 0.10 pools/day in controls). After 1 month of 750 mg/day of chenodeoxycholic acid, the FCR and PR of both VLDL and LDL apoB became normal while total plasma cholesterol increased significantly to 145 +/- 18 mg/dl. In a second patient who had been receiving 750 mg/day of chenodeoxycholic acid for 6 months lipoprotein kinetics were normal. These parameters did not change when the subject was switched to 750 mg/day ursodeoxycholic acid. We postulate that cholesterol biosynthesis in CTX is derepressed by a diminished hepatic pool of chenodeoxycholic acid and that the elevated secretion of apoB is a response to the increased rate of cholesterol production.     

Biosynthesis of a novel bile acid nucleotide and mechanism of 7 alpha-dehydroxylation by an intestinal Eubacterium species

Eubacterium species V.P.I. 12708 has inducible bile acid 7-dehydroxylase activity that can use either 7 alpha or 7 beta bile acids as substrates. Cell extracts prepared from bacteria grown in the presence of cholic acid catalyzed the rapid conversion of free bile acids into a highly polar bile acid metabolite (HPBA). This conjugation activity co-eluted with bile acid 7-dehydroxylase activity on high performance gel filtration chromatography (GFC). The HPBA was purified by a combination of high performance GFC and reverse-phase high performance liquid chromatography (HPLC). The intact HPBA eluted earlier from reverse-phase HPLC than deoxycholyl-CoA and had a Mr of 1102 by Bio-Gel P-2 (GFC). The HPBA had an absorption peak at 255 nm and was sensitive to treatment with phosphodiesterase I or nucleotide pyrophosphatase. The HPBA has a free phosphate as shown by an increase in elution volume on reverse-phase HPLC following treatment with alkaline phosphatase. Treatment of the purified HPBA with nucleotide pyrophosphate plus alkaline phosphatase yielded adenosine, whereas, treatment with nucleotide pyrophosphatase alone generated 5',3'-ADP. A bile acid metabolite was also generated by nucleotide pyrophosphatase treatment. The bile acid metabolite had different chromatographic properties (HPLC and TLC) than the corresponding free bile acid. Gas liquid chromatography-mass spectrometry showed the bile acid metabolite to be 12 alpha-hydroxy-3-oxo-4-cholenoic acid. We hypothesize that the HPBA is an intermediate in 7-dehydroxylation and consists of this compound linked at the C-24 with an anhydride bond to the beta phosphate (5') of ADP-3'-phosphate. These results suggest a novel mechanism of bile acid 7 alpha/7 beta-dehydroxylation in Eubacterium sp. V.P.I. 12708.     

Identification of short side chain bile acids in urine of patients with cerebrotendinous xanthomatosis

Urine from patients with cerebrotendinous xanthomatosis (CTX) was found to contain a number of minor bile acids along with three major bile acids, 7-epicholic acid, norcholic acid, and cholic acid. The following minor bile acids were identified by combined gas-liquid chromatography-mass spectrometry: 7-ketobisnordeoxycholic acid; 12-ketobisnorchenodeoxycholic acid; 7-ketonordeoxycholic acid; 12-ketochenodeoxycholic acid; 7-ketodeoxycholic acid; 12-ketochendeoxycholic acid; bisnorcholic acid; allonorcholic acid; allocholic acid; 1 beta-hydroxybisnorcholic acid; 1 beta-hydroxynorcholic acid; 1 beta-hydroxycholic acid; 2 beta-hydroxybisnorcholic acid; 2 beta-hydroxy-norcholic acid; 2 beta-hydroxycholic acid. The presence of C22 and C23 bile acids in urine of the CTX patients suggests that bile alcohols having a hydroxyl group at C22 or C23 in the side chain may be further degraded to these bile acids.     

Multi-parametric neuroimaging evaluation of cerebrotendinous xanthomatosis and its correlation with neuropsychological presentations

Background

Cerebrotendinous xanthomatosis (CTX) is a rare genetic disorder. Recent studies show that brain damage in CTX patients extends beyond the abnormalities observed on conventional magnetic resonance imaging (MRI). We studied the MRI and ^{99 m}Tc-ethyl cysteinate dimer single photon emission computed tomography (SPECT) findings of CTX patients and made a correlation with the neuropsychological presentations.

Methods

Diffusion tensor imaging (DTI) and 3D T1-weighted images of five CTX patients were compared with 15 age-matched controls. Voxel-based morphometry (VBM) was use to delineate gray matter (GM) and white matter (WM) volume loss. Fractional anisotropy (FA), mean diffusivity (MD), and eigenvalues derived from DTI were used to detect WM changes and correlate with neuropsychological results. SPECT functional studies were used to correlate with GM changes.

Results

Cognitive results showed that aside from moderate mental retardation, the patient group performed worse in all cognitive domains. Despite the extensive GM atrophy pattern, the cerebellum, peri-Sylvian regions and parietal-occipital regions were correlated with SPECT results. WM atrophy located in the peri-dentate and left cerebral peduncle areas corresponded with changes in diffusion measures, while axial and radial diffusivity suggested both demyelinating and axonal changes. Changes in FA and MD were preceded by VBM in the corpus callosum and corona radiata. Cognitive results correlated with FA changes.

Conclusion

In CTX, GM atrophy affected the perfusion patterns. Changes in WM included atrophy, and axonal changes with demyelination. Disconnection of major fiber tracts among different cortical regions may contribute to cognitive impairment.

     

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.     

Transformation of 5 alpha-cholest-7-en-3 beta-ol to cholesterol and cholestanol in cerebrotendinous xanthomatosis

The metabolism of Delta(7)-cholestenol, cholesterol, and cholestanol was examined in a patient with cerebrotendinous xanthomatosis after intravenous pulse-labeling with a mixture of dl-[2-(14)C]mevalonate and stereospecific 3S,4S,3R,4R-[4-(3)H]mevalonate. Silver nitrate and reversed-phase thin-layer chromatography were used to purify the sterols isolated from the feces, and their identities were confirmed by gas-liquid chromatography-mass spectrometry. The specific activities were determined and plotted as a function of time. Isotope ratio measurements and specific activity decay curves showed that sterol synthesis proceeded in the following sequence: mevalonate, squalene, lanosterol, Delta(7)-cholestenol, cholesterol, cholestanol. Labeled cholesterol precursors might be advantageously used to measure changes in cholesterol synthesis because they appear to equilibrate rapidly and have very short turnover times.     

Isolation of 5 alpha-cholestane-3 beta, 7 alpha-diol from bile of patients with cerebrotendinous xanthomatosis. Inefficiency of this steroid as a precursor to cholestanol

Cerebrotendinous xanthomatosis is a rare, inherited disease characterized by defective bile acid biosynthesis as well as by accumulation of cholesterol and cholestanol. The mechanism behind the accumulation of cholestanol is unknown. Using combined gas chromatography-mass spectrometry, 5 alpha-cholestane-3 beta, 7 alpha-diol could be identified as a minor component in bile from two such patients. There were no significant amounts of this steroid in bile from control subjects. Most probably, the 5 alpha-cholestan-3 beta, 7 alpha-diol found is formed from 7 alpha-hydroxy-4-cholesten-3-one in the liver. 7 alpha-Hydroxy-1-cholesten-3-one, being a normal intermediate in bile acid biosynthesis, is known to accumulate in the liver and bile of patients with cerebrotendinous xanthomatosis, due to a defect of the mitochondrial 26-hydroxylase. The possibility was tested that (7 beta-3H)-labeled 5 alpha-cholestane-3 beta, 7 alpha-diol could be converted into cholestanol by a direct 7 alpha-dehydroxylation in the intestine. This conversion did not occur in rabbits, however, regardless of whether the labelled steroid was administered orally or intracoecally. It is concluded that 5 alpha-cholestane-3 beta, 7 alpha-diol is of little or no importance as a precursor to cholestanol in rabbits. Most probably, this is also the case in patients with cerebrotendinous xanthomatosis.     

Absolute configuration of pentahydroxy bile alcohols excreted by patients with cerebrotendinous xanthomatosis: a circular dichroism study

The absolute configurations of the C27 pentahydroxy bile alcohols present in bile and feces of two patients with cerebrotendinous xanthomatosis (CTX) were determined by circular dichroism (CD) spectroscopy. The CD spectra of 5beta-cholestane-3alpha,7alpha,12alpha,24alpha,25-pentol in the presence of Eu(fod)3 [tris(1,1,1,2,2,3,3-heptafluoro-7,7-dimethyloctane-4,6-dionato) europium (III)] exhibited a negative Cotton effect and was assigned to 24R absolute configuration. Conversely, 5beta-cholestane-3alpha,7alpha,12alpha,24beta,25-pentol showed a strong positive Cotton effect and was assigned the 24S configuration. These assignments were based upon comparison with a model compound, 5-cholestene-3beta,24(R),25-triol, whose single-crystal X-ray structure has been determined. The importance of these data is to establish a structural mechanism for the conversion of 5beta-cholestane-3alpha,7alpha,12alpha,24S,25-pentol rather than 5beta-cholestane-3alpha,7alpha,12alpha,24R,25-pentol into cholic acid in man as well as in animals.     

Identification of 5 β-cholestane-3 α, 7 α, 12 α, 25, 26-pentol in cerebrotendinous xanthomatosis

An unrecognized pentahydroxy bile alcohol has been isolated from the bile and feces of patients with cerebrotendinous xanthomatosis (CTX). Its structure, 5 β-cholestane-3 α, 7 α, 12 α, 25, 26-pentol has been deduced by spectroscopic methods and confirmed by comparison with a synthetic analog.     

Tauroconjugation of cholic acid stimulates 7 alpha-dehydroxylation by fecal bacteria.

We examined the effect of the type of cholic acid conjugation (taurine-conjugated, glycine-conjugated, or unconjugated cholic acid) on cholic acid 7 alpha-dehydroxylation by intestinal flora. Cholic acid 7 alpha-dehydroxylation in fecal cultures, in cultures of a defined limited flora consisting of a mixture of seven bacterial species isolated from the intestinal tract, and in a binary culture of a 7 alpha-dehydroxylating Clostridium species plus a cholic acid-deconjugating Bacteroides species was studied. We found that tauroconjugation of cholic acid significantly (P < 0.05) increased bacterial 7 alpha-dehydroxylation of cholic acid into deoxycholic acid from 34 to 55% in fecal cultures, from 45 to 60% in defined limited fecal cultures, and from 75 to 100% in binary cultures. Equimolar concentrations of free taurine did not stimulate 7 alpha-dehydroxylation in fecal cultures or in the defined limited flora, but free taurine did stimulate 7 alpha-dehydroxylation in the binary culture. In the binary culture of Clostridium species strain 9/1 plus Bacteroides species strain R1, the minimal flora capable of increased 7 alpha-dehydroxylation of taurocholic acid, strain R1 deconjugated taurine and rapidly reduced it to H2S. Bacteroides species strain R1 did not grow unless taurine or another appropriate reducible sulfur source was present. Clostridium species strain 9/1 did not grow or 7 alpha-dehydroxylate unless H2S or another source of reduced sulfur was present. We conclude that the increased 7 alpha-dehydroxylation of tauroconjugated cholic acid depends on the reduction of taurine to H2S, which is a necessary growth factor for the 7 alpha-dehydroxylating bacteria.