Detection of carriers of cerebrotendinous xanthomatosis

Patients suffering from cerebrotendinous xanthomatosis (an autosomal recessive inborn error of metabolism) can easily be distinguished from patients not suffering from this disease, as the first excrete large amounts of the bile alcohol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol, in urine, whereas the second do not. In order to find out, whether carriers of cerebrotendinous xanthomatosis can be detected in a biochemical way, we compared known carriers with controls. The urinary excretions of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol of both groups were practically absent and no selection of carriers with cerebrotendinous xanthomatosis could be made on that basis. When, however, carriers and non-carriers were subjected to cholestyramine treatment, by which endogenous bile acid synthesis was stimulated, the urinary excretion of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol in the carrier rose considerably, whereas this excretion remained essentially the same in the non-carriers. This test can be of value in the genetic counseling of carriers with cerebrotendinous xanthomatosis and helpful in the detection of newborn patients with cerebrotendinous xanthomatosis.     

A Biochemical Abnormality in Cerebrotendinous Xanthomatosis IMPAIRMENT OF BILE ACID BIOSYNTHESIS ASSOCIATED WITH INCOMPLETE DEGRADATION OF THE CHOLESTEROL SIDE CHAIN

Bile acid production in cerebrotendinous xanthomatosis (CTX) is subnormal, yet the activity of cholesterol 7alpha-hydroxylase, the rate-determining enzyme of bile acid synthesis, is elevated. To explain this discrepancy, bile acid precursors were sought in bile and feces of three CTX subjects. Over 10% of the total sterols excreted in bile and feces consisted of compounds more polar than cholesterol. Chromatographic analysis of the polar fractions in conjunction with gasliquid chromatography (GLC)-mass spectrometry indicated two major constituents, 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol and 5beta-cholestane-3alpha,7alpha,12alpha,24xi,25-pentol. After i.v. injection of [4-(14)C]cholesterol both bile alcohols were radioactive proving that they were derived from cholesterol. The accumulation of alcohols hydroxylated at C-25 and C-24,25 suggests that decreased bile acid synthesis in CTX results from impaired oxidation of the cholesterol side chain. This finding and the virtual absence of intermediates hydroxylated at C-26 indicate that current views of the major pathway of bile acid synthesis may require revision.     

Bile acid therapies applied to patients suffering from cerebrotendinous xanthomatosis

Patients suffering from cerebrotendinous xanthomatosis, an inborn error of metabolism in bile acid synthesis, were given oral treatment with chenodeoxycholic acid, ursodeoxycholic acid, cholic acid and taurocholic acid. The effectiveness of the different therapies was evaluated by measuring the urinary excretion of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol, which should decrease, when the administered bile acid is able to suppress endogenous bile acid synthesis. From the results it is concluded that chenodeoxycholic acid and cholic acid activate the bile acid negative feedback mechanism, contrary to ursodeoxycholic acid and taurine conjugated cholic acid. Either cholic acid or chenodeoxycholic acid are the therapies of choice for the treatment of cerebrotendinous xanthomatosis. For various reasons the use of cholic acid is especially recommended.     

Effects of acute changes of bile acid pool composition on biliary lipid secretion.

To elucidate the mechanism responsible for the bile acid-induced changes of biliary lipid secretion, we evaluated bile flow and biliary output of bile acids, cholesterol, phospholipids, and alkaline phosphatase activity in seven cholecystectomized subjects with a balloon occludable T-tube during two experimental periods: (a) depletion of the endogenous bile acid pool and (b) replacement of the pool by means of duodenal infusion with individual bile acids, such as deoxycholic (DCA), chenodeoxycholic (CDCA), cholic (CA), and ursodeoxycholic (UDCA) acids. Bile flow, cholesterol, and phospholipid output were linearly related to bile acid secretion in all experimental periods. During the replacement periods, the amount of cholesterol and phospholipids coupled to bile acids was significantly different (at 1% level at least) for each individual bile acid secreted; it was the highest during DCA secretion (slope value: 0.209 for cholesterol and 0.434 for phospholipids) followed, in the order, by CDCA (0.078 and 1.794), CA (0.044 and 0.127), and UDCA (0.030 and 0.122). The phospholipid to cholesterol ratio was higher during secretion of CA and UDCA as compared with DCA and CDCA. The secretion of CA seemed to stimulate a greater bile flow than the other bile acids did. The infusion of all bile acids, except UDCA, induced an increase of biliary alkaline phosphatase activity as compared with the values of the depletion period. The mean highest increase (13-fold the pretreatment value) was observed during DCA secretion followed by CDCA (fivefold) and CA (1.5-fold). These results would suggest that the physical chemical properties, namely the lipid-solubilizing capacity, of bile acids could directly contribute to the regulation of biliary lipid secretion. The observed changes in biliary alkaline phosphatase activity lend support to the view that bile acid-induced lipid secretion may be, at least in part, contributed by membrane solubilization.     

Urinary excretion of bile acids during acute administration in man

Six healthy subjects, 45-72 years old, received a 10-day feeding of 750 mg of two of the following bile acids: deoxycholate (DCA), chenodeoxycholate (CDCA), cholate (CA), hyodeoxycholate (HDCA), ursodeoxycholate (UDCA), and ursocholate (UCA). The urinary excretion of total bile acids was low during administration of lipophilic bile acids (DCA and CDCA), when serum levels show low postabsorption peaks. Instead, hydrophilic bile acids (UDCA and above all HDCA) were heavily excreted in the urine as sulphates and glucuronides, and serum levels reach high values. Only UCA, strongly hydrophilic, was predominantly excreted as unconjugated fractions. Thus, the physicochemical properties of bile acids (as measured by both the partition between octanol and water, and the water solubility) were factors that influenced the route of bile acid elimination from the body, whereas their conjugation was not always requested for urinary excretion.     

Effect of cholestyramine on bile acid pattern and synthesis during administration of ursodeoxycholic acid in man

BACKGROUND: Cholestyramine is the first-line treatment for cholestasis-induced pruritus and is prescribed along with ursodeoxycholic acid (UDCA) in patients with cholestatic liver diseases. Impairment of the intestinal absorption of endogenous hydrophobic bile acids by cholestyramine is well known. It is unclear, however, whether cholestyramine also impairs the absorption of the hydrophilic bile acid, UDCA, in man. AIMS: To study serum levels of UDCA and endogenous bile acids as well as endogenous bile acid synthesis during simultaneous or separate administration of UDCA and cholestyramine in vivo; and absorption of UDCA both in the presence and absence of its hydrophobic epimer, chenodeoxycholic acid (CDCA), by cholestyramine in vitro. PATIENTS AND METHODS: Five healthy subjects received UDCA (12.5 +/- 0.5 mg kg-1 daily) as a single dose for periods of 14 days with or without cholestyramine (4 g daily). Fasting serum levels of bile acids and of 7alpha-hydroxy-4-cholesten-3-one (alpha-HC), a measure of endogenous bile acid synthesis, were determined by gas chromatography and high pressure liquid chromatography, respectively. In vitro, bile acid solutions were incubated for 24 h in the presence or absence of cholestyramine, and bile acid concentrations were determined in the supernatant. RESULTS: Simultaneous administration of UDCA and cholestyramine in man led to a decrease of fasting serum levels of UDCA by 60% when compared to UDCA serum levels during administration of UDCA alone. In contrast, serum levels of endogenous bile acids were not affected and alpha-HC serum levels were found increased 2. 7-fold indicating stimulation of endogenous bile acid synthesis by cholestyramine. Administration of cholestyramine and UDCA at an interval of 5 h tended to diminish the effect of cholestyramine on UDCA serum levels. In vitro, conjugated and unconjugated UDCA were effectively bound by cholestyramine both in the presence and absence of hydrophobic bile acids. CONCLUSIONS: The results strongly support the recommendation to administer UDCA and cholestyramine at different times of day.     

Cerebrotendinous xanthomatosis: a defect in mitochondrial 26-hydroxylation required for normal biosynthesis of cholic acid.

Oxidation of side chain of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol was studied in a patient with cerebrotendinous xanthomatosis (CTX) and in control subjects, using various subcellular fractions of liver homogenate and a method based on isotope dilution-mass spectrometry. In the control, 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol was converted into 5 beta-cholestane-3 alpha,7 alpha,12 alpha,26-tetrol and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid by the mitochondrial fraction, and into 5 beta-cholestane-3 alpha,7 alpha,12 alpha,-25-tetrol by the microsomal fraction. In the CTX patient, liver mitochondria were completely devoid of 26-hydroxylase activity. The same mitochondrial fraction catalyzed 25-hydroxylation of vitamin D3. The microsomal fraction of liver of the subject with CTX contained more than 50-fold the normal amount of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol. The basic metabolid defect in CTX appears to be a lack of the mitochondrial 26-hydroxylase. The excretion in the bile of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 alpha,25-pentol observed in CTX patients may be secondary to the accumulation of the major substrate for the 26-hydroxylase, i. e., 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol, and exposure of this substrate to the normally less active microsomal 25-and 24-hydroxylases. It is concluded that the major pathway in the biosynthesis of cholic acid in human liver involves a mitochondrial C27-steroid 26-hydroxylation.     

Occurrence of cholesterol monohydrate crystals in gallbladder and hepatic bile in man: influence of bile acid treatment

The occurrence of cholesterol monohydrate crystals was examined and related to the degree of cholesterol saturation in gallbladder bile and hepatic bile of gallstone (GS) patients (n = 34), gallstone-free (GSF) subjects (n = 33) and GS patients treated with chenodeoxycholic acid (CDCA (n = 7) or ursodeoxycholic acid (UDCA) (n = 11) for 3 weeks prior to cholecystectomy. Twenty-five untreated GS patients (74%) and four UDCA-treated patients (40%) displayed cholesterol crystals in the gallbladder bile. Only two GSF subjects (6%) and none of the CDCA-treated patients had crystals. Half of the patients with crystals in the gallbladder bile had crystals also in the hepatic bile. Cholesterol saturation of the gallbladder bile was higher in GS (142 +/- 15%, mean +/- SEM) than in GSF patients (74 +/- 5%). Saturation was also higher in GS patients with crystals (157 +/- 20%) than in those without crystals (99 +/- 12%). Gallbladder bile was unsaturated in all CDCA- and UDCA-treated patients. The results underline the importance of the degree of cholesterol saturation for the formation of cholesterol crystals. The data also give further support to the concept that the mechanism for inducing gallstone dissolution is different for CDCA and UDCA.     

Capillary gas chromatographic determination of cholestanol/cholesterol ratio in biological fluids. Its potential usefulness for the follow-up of some liver diseases and its lack of specificity in diagnosing CTX (cerebrotendinous xanthomatosis)

The concentration ratios of cholestanol/cholesterol in biological materials (serum, cerebrospinal fluid and tendon biopsy) were determined using a capillary gas chromatographic method. The method was validated by gas chromatography-mass spectrometry. The ratio was determined in several groups of patients: (a) patients with cerebrotendinous xanthomatosis (in serum, cerebrospinal fluid and tendon biopsy), before and during chenodeoxycholic acid therapy, (b) patients receiving cholestyramine therapy (in serum), (c) patients suffering from various liver diseases (in serum) and (d) one patient before and after liver transplantation (in serum). It can be concluded that the cholestanol/cholesterol concentration ratio is a potentially useful parameter for monitoring liver diseases but is not specific for establishing the diagnosis of cerebrotendinous xanthomatosis.     

Density and chemical solubility of cholesteric biliary calculi by cholelytic preparations of bile acids (CHENO-USO FALK) in vitro

Solubility of concrements in the bile obtained during surgery was studied in vitro in the presence of a definite amount of bile acids (CDCA and UDCA). Experiments were performed with multiple cholesterol concrements obtained from 37 patients who underwent cholecystectomy. Depending on the appearance, size and density the concrements were grouped as concrements of the same size or generation, concrements of two sizes, and concrements of more than two sizes so that they could be divided into three generations. It was established that density of one-generation concrements resembling pearl granules was far greater as compared to that of the other varieties of cholesterol concrements. The solving action of UDCA appeared more potent than that of CDCA.     

A novel pathway for biosynthesis of cholestanol with 7 alpha-hydroxylated C27-steroids as intermediates, and its importance for the accumulation of cholestanol in cerebrotendinous xanthomatosis.

A mixture of 7 alpha-3H- and 4-14C-labeled cholesterol was administered intravenously to rats. Cholestanol with 20-30% lower ratio between 3H and 14C than in cholesterol could be isolated from different organs. In a healthy human control, cholestanol isolated from feces had a 3H/14C ratio which was 28% lower than in administered cholesterol. Cholesterol and coprostanol reisolated in these experiments had the same ratio between 3H and 14C as in the precursor. A previously unknown pathway for formation of cholestanol, involving 7 alpha-hydroxylated intermediates, may explain these results. Under normal conditions, this pathway is responsible for at most 30% of the cholestanol synthesized from cholesterol. Intravenous administration of the 7 alpha-3H- and 4-14C-labeled cholesterol to a patient with cerebrotendinous xanthomatosis (CTX) resulted in formation of cholestanol which had 70-75% lower 3H/14C ratio. It is concluded that the novel pathway involving 7 alpha-hydroxylated intermediates is accelerated in patients with CTX. This acceleration may contribute essentially to the accumulation of cholestanol, which is a predominant feature of this disease. 7 alpha-Hydroxycholesterol and 7 alpha-hydroxy-4-cholesten-3-one might be intermediates in the novel pathway to cholestanol. After intravenous administration of 7 beta-3H-labeled 7 alpha-hydroxycholesterol in a patient with CTX, significant amounts of 3H were incorporated into plasma and fecal cholestanol. Only small amounts of 7 alpha-hydroxycholesterol and 7 alpha-hydroxy-4-cholesten-3-one are excreted into the intestine, and we therefore conclude that the 7 alpha-dehydroxylation step mainly occurs in the liver. In CTX, the synthesis of cholestanol may be accelerated because the concentrations of 7 alpha-hydroxylated bile acid intermediates in the liver are increased. A possible mechanism for the conversion of a minor fraction of 7 alpha-hydroxycholesterol into cholestanol is suggested.     

An analysis of data on human hepatic bile. Relationship between main bile components,serum cholesterol and serum triglycerides

Hepatic bile samples were taken from the common duct during interval operations for gallstone disease, performed under standardized conditions. Prior to operation serum cholesterol and triglycerides levels were determined. The concentrations of Cholesterol (Chol), phospholipids (Lip P) and of the three major bile acids (BA) were determined in ninety-seven samples. The data were subjected to statistical analysis. A highly significant rank correlation was found between the Chol- and the Lip P molar fractions. The rank correlation between the Chol molar fraction and the BA/Lip P ratio was highly significantly negative. Reasons are given why the observed intraindividual differences are interpreted as reflecting interindividual changes. The conclusion is that just as in the animal model, a rise of Chol in human hepatic bile is accompanied by a decrease of the BA/Lip P ratio. A significant correlation was found between chenodeoxycholic acid (CDCA), and the ratio between the two other main bile acids (CA/DCA). High lithogenicity was associated with low CDCA- and high DCA values. In a mathematical representation valid in the sense of a rank correlation, the bile/serum Chol ratio rose with increasing DCA values combined with increasing absolute differences between the two other acids. Serum triglycerides were negatively correlated with the BA molar fraction and with the absolute concentration of BA. As a result there was an association between serum triglycerides and lithogenicity.     

The Metabolism of Cholestanol, Cholesterol, and Bile Acids in Cerebrotendinous Xanthomatosis

The metabolism of cholesterol and its 5-dihydro derivative, cholestanol, was investigated by means of sterol balance and isotope kinetic techniques in 3 subjects with cerebrotendinous xanthomatosis (CTX) and 11 other individuals. All subjects were hospitalized on a metabolic ward and were fed diets practically free of cholesterol and cholestanol. After the intravenous administration of [1,2-(3)H]cholestanol, the radioactive sterol was transported and esterified in plasma lipoproteins in an identical manner to cholesterol. In these short-term experiments, the specific activity-time curves of plasma cholestanol conformed to two-pool models in both the CTX and control groups. However, cholestanol plasma concentrations, total body miscible pools, and daily synthesis rates were two to five times greater in the CTX than control individuals. The short-term specific activity decay curves of plasma [4-(14)C]cholesterol also conformed to two-pool models in both groups. However, in the CTX subjects the decay was more rapid, and daily cholesterol synthesis was nearly double that of the control subjects. Plasma concentrations and the sizes of the rapidly turning over pool of exchangeable cholesterol were apparently small in the CTX subjects, and these measurements did not correlate with the large cholesterol deposits found in tendon and tuberous xanthomas.Despite active cholesterol synthesis, bile acid formation was subnormal in the CTX subjects. However, bile acid sequestration was accompanied by a rise in plasma cholestanol levels and greatly augmented fecal cholestanol outputs. In contrast, the administration of clofibrate lowered plasma cholesterol levels 50% and presumably reduced synthesis in the CTX subjects. Plasma cholesterol concentrations and fecal steroid excretion did not change significantly during this therapy.These findings indicate that the excessive tissue deposits of cholesterol and cholestanol that characterize CTX were associated with hyperactive neutral sterol synthesis. The demonstration of subnormal bile acid formation suggests that defective bile acid synthesis may predispose to the neutral sterol abnormalities.     

A 25-hydroxylation pathway of cholic acid biosynthesis in man and rat.

This paper describes a pathway of cholic acid synthesis, in man and in the rat, which involves 25-hydroxylated intermediates and is catalyzed by microsomal and soluble enzymes. The subcellular localization, stereospecificity, and other properties of the enzymes involved were studied with liver fractions of normolipidemic subjects, cerebrotendinous xanthomatosis patients, and rats. 5beta-Cholestane-3alpha,7alpha,12alpha,25-tetrol was converted to 5beta-cholestane-3alpha,7alpha,12alpha,24beta,25-pentol by the microsomal fraction in the presence of NADPH and O2. 5beta-Cholestane-3alpha,7alpha,12alpha,24alpha,25-pentol, 5beta-cholestane-3alpha,7alpha,12alpha,-23xi,25-pentol, and 5beta-cholestane-3alpha,7alpha,12alpha,25,26-pentol were also formed. In the presence of NAD, 5beta-cholestane-3alpha,7alpha,12alpha,24beta,25-pentol, but not the other 5beta-cholestanepentols formed, was converted to cholic acid by soluble enzymes in good yield. These experiments demonstrate the existence of a pathway for side-chain degradation in cholic acid synthesis which does not involve hydroxylation at C-26 or the participation of mitochondria.     

Bile acid N-acetylglucosaminidation. In vivo and in vitro evidence for a selective conjugation reaction of 7 beta-hydroxylated bile acids in humans.

The aim of this study was to define whether N-acetylglucosaminidation is a selective conjugation pathway of structurally related bile acids in humans. The following bile acids released enzymatically from N-acetylglucosaminides were identified: 3 alpha,7 beta-dihydroxy-5 beta-cholanoic (ursodeoxycholic), 3 beta, 7 beta-dihydroxy-5 beta-cholanoic (isoursodeoxycholic), 3 beta,7 beta-dihydroxy-5 alpha-cholanoic (alloisoursodeoxycholic), 3 beta,7 beta-dihydroxy-5-cholenoic, 3 alpha,7 beta,12 alpha-trihydroxy-5 beta-cholanoic, and 3 alpha,6 alpha,7 beta-trihydroxy-5 beta-cholanoic acids. The selectivity of conjugation was studied by administration of 0.5 g ursodeoxycholic (UDCA) or hyodeoxycholic (HDCA) acids, labeled with 13C, to patients with extrahepatic cholestasis, and of 0.5 g of 13C-labeled chenodeoxycholic acid (CDCA) to patients with extra- or intrahepatic cholestasis. After administration of [24-13C]-CDCA, labeled glucosides, and the glucuronide of CDCA were excreted in similar amounts. Labeled N-acetylglucosaminides of UDCA and isoUDCA were also formed. When [24-13C]-UDCA was given, 13C-label was detected in the N-acetylglucosaminide, the glucosides, and the glucuronide of UDCA, and in the N-acetylglucosaminide of isoUDCA. In the patient studied, 32% of the total UDCA excreted in urine was conjugated with N-acetylglucosamine. In contrast, 96% of the excreted amount of [24-13C]HDCA was glucuronidated, and 13C-labeled glucosides but no N-acetylglucosaminide were detected. The selectivity of N-acetylglucosaminidation towards bile acids containing a 7 beta-hydroxyl group was confirmed in vitro using human liver and kidney microsomes and uridine diphosphate glucose (UDP)-N-acetylglucosamine. These studies show that N-acetylglucosaminidation is a selective conjugation pathway for 7 beta-hydroxylated bile acids.     

Reduction of urinary bile alcohol excretion and serum cholestanol in patients with cerebrotendinous xanthomatosis after oral administration of deoxycholic acid

Deoxycholic acid and chenodeoxycholic acid were administered alternately to four patients with cerebrotendinous xanthomatosis. During this oral therapy serum cholestanol and urinary bile alcohols were determined. Both showed a marked decrease after the start of the two different therapies. It can be concluded that not only chenodeoxycholic acid but also deoxycholic acid is able to suppress endogenous human bile acid synthesis, which is in accordance with other experiments describing the effect of feeding of various bile acids on endogenous bile acid synthesis.     

Role of the 26-hydroxylase in the biosynthesis of bile acids in the normal state and in cerebrotendinous xanthomatosis. An in vivo study.

On the basis of different in vitro studies, we have previously suggested that the basic metabolic defect in the rare inherited disease cerebrotendinous xanthomatosis (CTX) is a lack of a hepatic mitochondrial C27-steroid 26-hydroxylase, involved in the normal biosynthesis of bile acids (1980. J. Clin. Invest. 65: 1418-1430; 1981. J. Lipid Res. 22: 191-200; 22: 632-640). In the present work, this hypothesis was tested in vivo. One patient with CTX and two control subjects received intravenously a mixture of [4-14C]7 alpha-hydroxy-4-cholesten-3-one and [6 beta-3H]7 alpha,26-dihydroxy-4-cholesten-3-one, steroids believed to be important precursors of chenodeoxycholic acid. The ratio between 14C and 3H in cholic acid and chenodeoxycholic acid isolated from bile of the CTX-patient was approximately 1/40 and 1/60 of those of the control subjects, respectively. Another patient with CTX and one control subject received a mixture of [4-14C]5 beta-cholestane-3 alpha,7 alpha-diol and [1,2-3H]5 beta-cholestane-3 alpha,7 alpha,26-triol, both possible precursors to chenodeoxycholic acid. In this case the 14C/3H ratio in cholic acid and chenodeoxycholic acid from the patient with CTX was 1/10 and 1/15, respectively, compared with that of the control subject. The most likely explanation for these findings is that very little of the 14C-precursors, i.e. without a 26-hydroxyl group, can be converted into cholic acid and chenodeoxycholic acid because of a defect of the 26-hydroxylase step. The results obtained are in accord with our previous findings in vitro. The results further underline the importance of the 26-hydroxylase pathway in the normal biosynthesis of cholic acid and chenodeoxycholic acid in man.     

胆汁酸谱在肺炎和肺癌鉴别诊断中的应用价值

目的 探讨肺癌患者血清胆汁酸谱的变化及其在肺炎与肺癌鉴别诊断中的价值.方法 采用液相色谱-串联质谱法(LC-MS/MS)检测80例肺炎患者(肺炎组)、108例肺癌患者(肺癌组)和106名体检健康者(正常对照组)血清胆汁酸谱[5种游离胆汁酸,包括胆酸(CA)、鹅脱氧胆酸(CDCA)、脱氧胆酸(DCA)、石胆酸(LCA)、...     

Bile alcohol metabolism in man. Conversion of 5beta-cholestane-3alpha, 7alpha,12alpha, 25-tetrol to cholic acid.

To study the role of C25-HYDROXY BILE ALCOHOLS AS PRECURSORS OF CHOlic acid, [G-3-H]5beta-cholestane-3alpha,7alpha12alpha,25-tetrol was administered intravenously to two subjects with cerebrotendinous xanthomatosis (CTX) and two normal individuals. One day after pulse labeling, radioactivity was present in the cholic acid isolated from the bile and feces of the subjects with CTX and the bile of the normal individuals. In the two normal subjects, the sp act decay curves of [G-3-H]-cholic acid were exponential, and no traces of [G-3-H]-5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol were detected. In contrast, appreciable quantities of labeled 5beta-cholestane-3alpha,-7aopha,12alpha,25-tetrol were present in the bile and feces of the CTX subjects. The sp act vs. time curves of fecal [G-3-H]5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol and [G-3-H]-cholic acid showed a precursor-product relationship. Although these results suggest that 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol may be a precursor of cholic acid in man, the possibility that C26-hydroxy intermediates represent the normal pathway can not be excluded.     

Biosynthesis of bile acids in cerebrotendinous xanthomatosis. Relationship of bile acid pool sizes and synthesis rates to hydroxylations at C-12, C-25, and C-26.

To examine the defect in side-chain oxidation during the formation of bile acids in cerebrotendinous xanthomatosis, we measured in vitro hepatic microsomal hydroxylations at C-12 and C-25 and mitochondrial hydroxylation at C-26 and related them to the pool size and synthesis rates of cholic acid and chenodeoxycholic acid as determined by the isotope dilution technique. Hepatic microsomes and mitochondria were prepared from seven subjects with cerebrotendinous xanthomatosis and five controls. Primary bile acid synthesis was markedly reduced in cerebrotendinous xanthomatosis as follows: cholic acid, 133 +/- 30 vs. 260 +/- 60 mg/d in controls; and chenodeoxycholic acid, 22 +/- 10 vs. 150 +/- 30 mg/d in controls. As postulated for chenodeoxycholic acid synthesis, mitochondrial 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol was present in all specimens and was 30-fold more active than the corresponding microsomal 25-hydroxylation. However, mean mitochondrial 26-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha-diol was less active in cerebrotendinous xanthomatosis than in controls: 59 +/- 17 compared with 126 +/- 21 pmol/mg protein per min. As for cholic acid synthesis, microsomal 25-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol was substantially higher in cerebrotendinous xanthomatosis and control preparations (620 +/- 103 and 515 +/- 64 pmol/mg protein per min, respectively) than the corresponding control mitochondrial 26-hydroxylation of the same substrate (165 +/- 25 pmol/mg protein per min). Moreover in cerebrotendinous xanthomatosis, mitochondrial 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol-26-hydroxylase activity was one-seventh as great as in controls. Hepatic microsomal 12 alpha-hydroxylation, which may be rate-controlling for the cholic acid pathway, was three times more active in cerebrotendinous xanthomatosis than in controls: 1,600 vs. 500 pmol/mg protein per min. These results demonstrate severely depressed primary bile acid synthesis in cerebrotendinous xanthomatosis with a reduction in chenodeoxycholic acid formation and pool size disproportionately greater than that for cholic acid. The deficiency of chenodeoxycholic acid can be accounted for by hyperactive microsomal 12 alpha-hydroxylation that diverts precursors into the cholic acid pathway combined with decreased side-chain oxidation (mitochondrial 26-hydroxylation). However, side-chain oxidation in cholic acid biosynthesis may be initiated via microsomal 25-hydroxylation of 5beta-cholestane-3alpha,7alpha,12alpha-triol was substantially lower in control and cerebrotendinous xanthomatosis liver. Thus, separate mechanisms may exist for the cleavage of the cholesterol side chain in cholic acid and chenodeoxycholic acid biosynthesis.