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The accumulation of various 25-hydroxylated C 27 -bile alcohols in blood and their excretion in urine are characteristic features of cerebrotendinous xanthomatosis (CTX) a recessively inherited inborn error of bile acid synthesis caused by mutations in the mitochondrial sterol 27-hydroxylase (CYP27) gene. These bile alcohols may be intermediates in the alternative cholic acid side chain cleavage pathway. The present study was undertaken to identify enzymes and reactions responsible for the formation of these bile alcohols and to explain why Cyp27 ؊/؊ mice do not show CTX-related abnormalities. Microsomal activities of 5␤-cholestane-3␣,7␣,12␣-triol 25-and 26-hydroxylases, 5␤-cholestane-3␣,7␣,12␣,25-tetrol 23R-, 24S-, and 27-hydroxylases and testosterone 6␤-hydroxylase, a marker enzyme for CYP3A, in Cyp27 ؊/؊ mice livers were markedly up-regulated (5.5-, 3.5-, 6.5-, 7.5-, 2.9-, and 5.4-fold, respectively). In contrast, these enzyme activities were not increased in CTX. The activities of 5␤-cholestane-3␣,7␣,12␣-triol 25-and 26-hydroxylases and 5␤-cholestane-3␣,7␣,12␣,25-tetrol 23R-, 24R-, 24S-, and 27-hydroxylases were strongly correlated with the activities of testosterone 6␤-hydroxylase in control human liver microsomes from eight unrelated donors. Troleandomycin, a specific inhibitor of CYP3A, markedly suppressed these microsomal side chain hydroxylations in both mouse and human livers in a dosedependent manner. In addition, experiments using recombinant overexpressed human CYP3A4 confirmed that these microsomal side chain hydroxylations were catalyzed by a single enzyme, CYP3A4. The results demonstrate that microsomal 25-and 26-hydroxylations of 5␤-cholestane-3␣,7␣,12␣-triol and microsomal 23R-, 24R-, 24S-, and 27-hydroxylations of 5␤-cholestane-3␣,7␣,12␣,25-tetrol are mainly catalyzed by CYP3A in both mice and humans. Unlike Cyp27 ؊/؊ mice, CYP3A activity was not up-regulated despite marked accumulation of 5␤-cholestane-3␣,7␣,12␣-triol in CTX.

Using plasma isotope-kinetic methods, we measured the absorption and turnover rates of cholesterol and sitosterol (24-ethylcholesterol) in two obligate heterozygotes (parents) and their homozygous daughter with sitosterolemia with xanthomatosis. Diets contained approximately 500 mg/day cholesterol and 100 mg/day sitosterol. In the homozygote, plasma cholesterol and apolipoprotein B concentrations were slightly higher, but sitosterol levels were 22 and 58 times higher than in her heterozygous parents. Cholesterol absorption was at the high end of the normal range in both heterozygotes (59% and 84%) and in the homozygote (62%) (value in the control subject 48%). In contrast, cholesterol synthesis was severely depressed in the homozygote (28% and 26% as great as in the heterozygotes and the control, respectively). Sitosterol absorption in the homozygote (34%) was 12 and 2.0 times greater than in the heterozygotes and 6.8 times greater than in the control. The sitosterol turnover rate, calculated independently by mathematical analysis of specific-activity decay curves, amounted to 15 and 24 mg/day in the heterozygotes compared with 27 mg/day in the homozygote and 7.9±2J mg/day in five control subjects. However, the total body sitosterol pool was 15 and 103 times larger in the homozygote (4,080 mg) than in her heterozygous parents because of extremely slow removal. The average sitosterol elimination constant in the heterozygotes (K A =0.11 day" 1 ) was 10 times that in the homozygote (K A =0.01 day" 1 ) but 35% less than that in the controls (K A =0.17 day" 1 ). These results demonstrate that despite enhanced sitosterol absorption, small body pools and low plasma concentrations result from rapid elimination associated with adequate cholesterol synthesis in sitosterolemic heterozygotes. In distinction, sitosterol accumulates and body pools are disproportionately enlarged because increased absorption is combined with decreased removal, which may compensate for reduced cholesterol synthesis in sitosterolemic homozygotes. S itosterolemia with xanthomatosis is a rare lipid storage disease that presents clinically with accelerated atherosclerosis, tendon and tuberous xanthomas, hemotysis, and symmetrical arthritis and arthralgias involving the ankle and knee joints.1 Increased amounts of plant sterols (sitosterol, campesterol, and stigmasterol) and 5a-stanols (cholestanol, 5a-sitostanol, and 5a-campestanol) are present in plasma and all tissues except brain.2 Enhanced absorption of sitosterol and structurally related sterols has been suggested to account for the enlarged pools, 3 -7 while cholestanol and 5o-saturated plant sterol derivatives are produced endogenousry because diets are virtually devoid of these stanols. 8 - 10 The disease isFrom the Veterans Administration Medical Center, East Orange, and the University of Medicine and Dentistry-New Jersey Medical College, Newark, NJ.Supported by Research Service, Veterans Administration, and US Public Health Service grants HL-17818, DK-18707, and DK-26756.Address for c...

We examined the relationship between cholesterol biosynthesis and total and high affinity LDL binding in liver specimens from two sitosterolemic and 12 healthy control subjects who died unexpectedly and whose livers became available when no suitable recipient for transplantation was identified. Accelerated atherosclerosis, unrestricted intestinal sterol absorption, increased plasma and tissue plant sterol concentrations, and low cholesterol synthesis characterize this disease. Mean total microsomal HMG-CoA reductase (rate-control controlling enzyme for cholesterol biosynthesis) activity was sevenfold higher (98.1±28.8 vs. 15.0±2.0 pmol/mg protein per min) and microsomal enzyme protein mass was eightfold larger (1.43±0.41 vs. 0.18±0.04 relative densitometric U/mg protein) in 11 controls than the average for two sitosterolemic liver specimens. HMG-CoA reductase mRNA probed with pRED 227 and pHRED 102 was decreased to barely detectable levels in the sitosterolemic livers. In addition, there was a 50% decrease in the rate 12-'4Cimevalonic acid was converted to cholesterol by sitosterolemic liver slices compared with controls (112 vs. 224±32 pmol/g liver per h). In contrast, average total LDL binding was 60% greater (326 vs. 204±10 ng/mg), and high affinity (receptor-mediated) binding 165% more active (253 vs. 95.1±8.2 ng/mg) in two sitosterolemic liver membrane specimens than the mean for 12 controls. Liver morphology was intact although sitosterolemic hepatocytes and microsomes contained 24 and 14% less cholesterol, respectively, and 10-100 times more plant sterols and 5a-stanols than control specimens.We postulate that inadequate cholesterol biosynthesis is an inherited abnormality in sitosterolemia and may be offset by augmented receptor-mediated LDL catabolism to supply cellular sterols that cannot be formed. (J. Clin. Invest. 1990. 86:923-931.)

We investigated the effect of chenodeoxycholic acid on cerebrospinal fluid sterol and protein composition in six patients with cerebrotendinous xanthomatosis, a progressive neurologic disease, and in 11 control subjects. In the cerebrospinal fluid from the controls, the mean (+/- SD) levels of cholesterol and cholestanol were 400 +/- 300 and 4 +/- 7 micrograms per deciliter, respectively. The levels were almost 1.5 and 20 times higher in cerebrospinal fluid from untreated patients with cerebrotendinous xanthomatosis. Cholestanol levels were also markedly elevated in the plasma of untreated patients, but their plasma cholesterol levels (215 +/- 61 mg per deciliter) were not different from control values. Treatment with chenodeoxycholic acid reduced cerebrospinal fluid cholesterol by 34 percent and cholestanol threefold. Plasma cholestanol levels also decreased sharply. Normal cerebrospinal fluid contained small quantities of albumin, apolipoproteins, and lecithin:cholesterol acyltransferase. In cerebrospinal fluid from untreated patients with cerebrotendinous xanthomatosis, immunoreactive apolipoprotein B or apolipoprotein B fragment was increased about 100-fold and albumin about 3.5-fold; apolipoprotein AI, apolipoprotein D, and lecithin:cholesterol acyltransferase were 1.5 to 3 times more concentrated. Apolipoprotein AIV and apolipoprotein E concentrations were comparable to those in controls, and apolipoprotein AII was considerably decreased. During treatment, the concentrations of albumin and apolipoproteins AI and B declined. These results suggest that increased cerebrospinal fluid sterols are derived from plasma lipoproteins by means of a defective blood-brain barrier in patients with cerebrotendinous xanthomatosis. Therapy with chenodeoxycholic acid reestablished selective permeability of the blood-brain barrier and normalized the concentrations of sterol and apolipoprotein in the cerebrospinal fluid.

The regulation of the rabbit apical sodium-dependent bile acid transporter (ASBT) was studied both in vivo and in vitro. New Zealand White rabbits were fed 0.5% deoxycholic acid (DCA) or SC-435, a competitive ASBT inhibitor, for 1 wk. In DCA-fed rabbits, ASBT expression was repressed, associated with activated FXR, and evidenced by increased ileal short heterodimer partner (SHP) mRNA. Feeding SC-435 to the rabbits blocked bile acid absorption, decreased SHP mRNA, and increased ASBT expression. A 1.9-kb rabbit ASBT 5Ј-flanking region (promoter) was cloned, and a cis-acting element for ␣-fetoprotein transcription factor (FTF) was identified (Ϫ1166/ Ϫ1158). The effects of transcriptional factors and different bile acids on the rabbit ASBT promoter were studied in Caco-2 cells. FTF stimulated the rabbit ASBT promoter activity fourfold but not after the FTF binding site was deleted from the promoter. Increasing the SHP protein notably inhibited FTF-dependent trans-activation of rabbit ASBT. Adding hydrophobic bile acids deoxycholic acid, chenodeoxycholic acid, and cholic acid, activating ligands for FXR, inhibited rabbit ASBT promoter activity in Caco-2 cells, but this inhibitory effect was abolished after the FTF binding site was deleted. Ursodeoxycholic acid and ursocholic acid, nonactivating ligands for FXR, did not repress ASBT promoter activity. Thus the rabbit ASBT promoter is negative-feedback regulated by bile acids via a functional FTF binding site. Only FXR-activating ligands can downregulate rabbit ASBT expression through the regulatory cascade FXR-SHP-FTF.THE APICAL SODIUM-DEPENDANT bile acid cotransporter (ASBT/ SLC10A2) is the primary bile salt uptake protein in the intestine. It is mainly located on the apical surface of the terminal ileal enterocytes and is also expressed on renal proximal tubular cells and large cholangiocytes (12,19). ASBT is an efficient transporter for conjugated and unconjugated bile salts. Bile salt reabsorption by ASBT in the ileum is sodium dependent and can be saturated (7). ASBT has been cloned from the human (26), rabbit (11), rat (19), mouse (17), and hamster (25).Regulation of ASBT expression by intestinal bile acid flux has been studied in guinea pigs (13), rats (2,8,10,18,20), and mice (21). However, whether ASBT expression is positively or negatively regulated by increasing bile acid flux remains controversial. Observations in guinea pigs (13) and mice (21) showed that ASBT was negatively regulated by the intestinal bile acid flux, whereas in rats, ASBT was positively regulated by bile acids (8,10,18,20). Nevertheless, the results reported by Arrese et al. (2) showed that in rats, no regulatory response to changes in the intestinal bile acid flux occurred. Recently, Chen et al. (5) identified a physiologically functional liver receptor homolog-1 (LRH-1; also called FTF, ␣-fetoprotein transcription factor in other species) transcriptional binding site in the mouse ASBT promoter that was not present in the rat. As a result, chenodeoxycholic acid (CDCA), an activating lig...