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Cerebrotendinous xanthomatosis (CTX) is a rare, recessively inherited lipid storage disease characterized by a markedly reduced production of chenodeoxycholic acid and an increased formation of 25-hydroxylated bile alcohols and cholestanol. Patients with this disease are known to have mutations in the sterol 27-hydroxylase (Cyp27) gene. However, one study showed that mice with a disrupted Cyp27 gene did not have any CTX-related clinical or biochemical abnormalities. To explore the reason, hepatic cholesterol, cholestanol, and 12 intermediates in bile acid biosynthetic pathways were quantified in 10 Cyp27(-/-) and 7 Cyp27(+/+) mice, two CTX patients (untreated and treated with chenodeoxycholic acid), and four human control subjects by high resolution gas chromatography-mass spectrometry. Mitochondrial 27-hydroxycholesterol and 5beta-cholestane-3alpha,7alpha,12alpha,27-tetrol were virtually absent in both Cyp27(-/-) mice and CTX patients. In Cyp27(-/-) mice, microsomal concentrations of intermediates in the early bile acid biosynthetic pathway (7alpha-hydroxycholesterol, 7alpha-hydroxy-4-cholesten-3-one, 7alpha,12alpha-dihydroxy-4-cholesten-3-one, and 5beta-cholestane-3alpha,7alpha,12alpha-triol), 25-hydroxylated bile alcohols (5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol, 5beta-cholestane-3alpha,7alpha,12alpha,23R,25-pentol, and 5beta-cholestane-3alpha,7alpha,12alpha,24R, 25-pentol), and cholestanol were all significantly elevated compared with those in Cyp27(+/+) mice, although the levels were lower than those in untreated CTX patients. The intermediate levels in early bile acid biosynthesis were more elevated in male (16;-86% of CTX) than in female Cyp27(-/-) mice (7-30% of CTX). In contrast, 25-hydroxylated bile alcohol concentrations were not significantly different between male and female Cyp27(-/-) mice and were considerably lower (less than 14%) than those in CTX patients.These results suggest that 1) in Cyp27(-/-) mice, especially in females, classic bile acid biosynthesis via 7alpha-hydroxycholesterol is not stimulated as much as in CTX patients; and 2) formed 25-hydroxylated bile alcohols are more efficiently metabolized in Cyp27(-/-) mice than in CTX patients.

Sterol 27-hydroxylase is important for the degradation of the steroid side chain in conversion of cholesterol into bile acids and has been ascribed a regulatory role in cholesterol homeostasis. Its deficiency causes the autosomal recessive disease cerebrotendinous xanthomatosis (CTX), characterized by progressive dementia, xanthomatosis, and accelerated atherosclerosis. Mice with a disrupted cyp27 (cyp27(-/-)) had normal plasma levels of cholesterol, retinol, tocopherol, and 1,25-dihydroxyvitamin D. Excretion of fecal bile acids was decreased (<20% of normal), and formation of bile acids from tritium-labeled 7alpha-hydroxycholesterol was less than 15% of normal. Compensatory up-regulation of hepatic cholesterol 7alpha-hydroxylase and hydroxymethylglutaryl-CoA reductase (9- and 2-3-fold increases in mRNA levels, respectively) was found. No CTX-related pathological abnormalities were observed. In CTX, there is an increased formation of 25-hydroxylated bile alcohols and cholestanol. In bile and feces of the cyp27(-/-) mice only traces of bile alcohols were found, and there was no cholestanol accumulation. It is evident that sterol 27-hydroxylase is more important for bile acid synthesis in mice than in humans. The results do not support the contention that 27-hydroxylated steroids are critical for maintenance of cholesterol homeostasis or levels of vitamin D metabolites in the circulation.

Cultured macrophages and endothelial cells have been reported to secrete 27-oxygenated metabolites of cholesterol. This mechanism was compared with the classical high density lipoprotein (HDL)-dependent reverse cholesterol transport. Under standard conditions, macrophage preparations had considerably higher capacity to secrete 27-hydroxycholesterol and 3beta-hydroxy-5-cholestenoic acid than had endothelial cells and fibroblasts. Western blotting showed that lung macrophages contained the most sterol 27-hydroxylase protein of the cells tested. The relative amounts of 3beta-hydroxy-5-cholestenoic acid produced by the macrophages were also highest. Macrophages derived from monocytes of patients with sterol 27-hydroxylase deficiency did not secrete 27-oxygenated products, demonstrating that sterol 27-hydroxylase is the critical enzyme for the conversion of cholesterol into the 27-oxygenated steroids. That sterol 27-hydroxylase is responsible not only for 27-hydroxylation of cholesterol but also for the further oxidation of this steroid into 3beta-hydroxy-5-cholestenoic acid was shown with use of tritium-labeled 27-hydroxycholesterol and an inhibitor of sterol 27-hydroxylase. Secretion of 27-oxygenated products by the cultured macrophages as well as the ratio between the alcohol and the acid appeared to be dependent upon total 27-hydroxylase activity, the availability of substrate cholesterol, and the presence of an acceptor for 27-hydroxycholesterol in the medium. With albumin as extracellular acceptor, the major secreted product was 3beta-hydroxy-5-cholestenoic acid. Under such conditions, secretion of labeled 27-oxygenated products was higher than that of labeled cholesterol from lung alveolar macrophages preloaded with [4-14C]cholesterol. With HDL as acceptor, 27-hydroxycholesterol was the major secreted product, and the total secretion of labeled 27-oxygenated products was only about 10% of that of labeled cholesterol. Thus, 27-hydroxycholesterol and cholesterol may compete for HDL-mediated efflux from the cells. The results support the contention that the sterol 27-hydroxylase-mediated elimination of cholesterol is more important in macrophages than in endothelial cells. This mechanism may be an alternative and/or a complement to the classical HDL-mediated reverse cholesterol transport in macrophages, in particular when the concentration of HDL is low.

The ligand-binding domain of the low density lipoprotein (LDL) receptor is composed of seven cysteine-rich repeats, each approximately 40 amino acids long. Previous studies by van Driel et al. [van Driel, I. R., Goldstein, J. L., Sudhof, T. C. & Brown, M. S. (1987) J. Biol. Chem. 262, 17443-17449] showed that if the first repeat of the ligand-binding domain (encoded by exon 2) is deleted, the receptor fails to bind an anti-LDL receptor monoclonal antibody (IgG-C7) but continues to bind LDL with high affinity. Cultured fibroblasts from a Black South African Xhosa patient (TT) with the clinical syndrome of homozygous familial hypercholesterolemia demonstrated high-affinity cell-surface binding of 125I-labeled LDL but not 125I-labeled IgG-C7. Previous haplotype analysis, using 10 restriction fragment length polymorphic sites, suggested that the patient inherited two identical LDL receptor alleles. The polymerase chain reaction technique was used to selectively amplify exon 2 of the LDL receptor gene from this patient. Sequence analysis of the amplified fragment disclosed a deletion of six base pairs that removes two amino acids, aspartic acid and glycine, from the first cysteine-rich ligand binding repeat. The mutation creates a new PstI restriction site that can be used to detect the deletion. The existence of this mutant allele confirms that the epitope of IgG-C7 is located in the first cysteine-rich repeat and that this repeat is not necessary for LDL binding. The mutant gene produced a normally sized 120-kilodalton LDL receptor precursor protein that matured to the 160-kilodalton form at less than one-fourth the normal rate. Thus, deletion of two amino acids within the first cysteine-rich repeat retards receptor transport from the endoplasmic reticulum to the cell surface, in contrast to deletion of the entire first repeat, which has no effect on receptor maturation.