Abstract
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.
Highlights
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
It is evident that sterol 27-hydroxylase is more important for bile acid synthesis in mice than in humans
The enzyme is important for bile acid biosynthesis but has been ascribed a role in connection with cholesterol removal from extrahepatic tissues, in regulation of cholesterol homeostasis, and in metabolism of vitamin D
Summary
Cloning of the Mouse cyp and Construction of the Targeting Plasmid—Oligonucleotides complementary to the putative exon 3 of the rat cyp were prepared (24 –26). To verify the planned disruption of the gene, a probe from the putative mouse exon 3 was prepared and used in Southern blotting to identify a 5- and a 16-kb XbaI fragment from the targeted and non-targeted alleles, respectively (Fig. 2a). In a third step the sections were stained in a picric acid/thiazine red mixture (10:0.2) for 10 min, rinsed in distilled water containing picric acid, and dehydrated and coverslipped by an automated coverslipper (Sakura). The whole aorta was fixed in 10% buffered formalin, rinsed in tap water, cut open, blocked with needles, and stained with Sudan III. Oxysterols (7␣-hydroxycholesterol, 24-hydroxycholesterol, and 27-hydroxycholesterol in serum and in various organs) were analyzed by isotope dilution-mass spectrometry with use of deuterium-labeled internal standards as described previously [40]. The amount of cholic acid had been quantitated by isotope dilution-mass spectrometry in a separate analysis as described above. Radioactivity was measured in aliquots of the hexane phase containing neutral steroids and of the ether phase containing bile acids
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