Regulation of 25- and 27-hydroxylation side chain cleavage pathways for cholic acid biosynthesis in humans, rabbits, and mice. Assay of enzyme activities by high-resolution gas chromatography;-mass spectrometry.
In classic cholic acid biosynthesis, a series of ring modifications of cholesterol precede side chain cleavage and yield 5beta-cholestane-3alpha, 7alpha, 12alpha-triol. Side chain reactions of the triol then proceed either by the mitochondrial 27-hydroxylation pathway or by the microsomal 25-hydroxylation pathway. We have developed specific and precise assay methods to measure the activities of key enzymes in both pathways, 5beta-cholestane-3alpha, 7alpha, 12alpha-triol 25- and 27-hydroxylases and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases. The extracts from either the mitochondrial or microsomal incubation mixtures were purified by means of a disposable silica cartridge column, derivatized into trimethylsilyl ethers, and quantified by gas chromatography;-mass spectrometry with selected-ion monitoring in a high resolution mode. Compared with the addition of substrates in acetone, those in 2-hydroxypropyl-beta-cyclodextrin increased mitochondrial triol 27-hydroxylase activity 132% but decreased activities of the enzymes in microsomal 25-hydroxylation pathway (triol 25-hydroxylase and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases) 13;-60% in human liver. The enzyme activities in both pathways were generally 2- to 4-times higher in mouse and rabbit livers compared with human liver. In all species, microsomal triol 25-hydroxylase activities were 4- to 11-times larger than mitochondrial triol 27-hydroxylase activities but the activities of tetrol 24S-hydroxylase were similar to triol 27-hydroxylase activities in our assay conditions. The regulation of both pathways in rabbit liver was studied after bile acid synthesis was perturbed. Cholesterol feeding up-regulated enzyme activities involved in both 25- (64;-142%) and 27- (77%) hydroxylation pathways, while bile drainage up-regulated only the enzymes in the 25-hydroxylation pathway (178;-371%). Using these new assays, we demonstrated that the 25- and 27-hydroxylation pathways for cholic acid biosynthesis are more active in mouse and rabbit than human livers and are separately regulated in rabbit liver.
Honda
A
A
Department of Gastroenterology, University of Tsukuba, Tsukuba-city 305-8575, Japan.
Salen
G
G
Shefer
S
S
Matsuzaki
Y
Y
Xu
G
G
Batta
A K
AK
Tint
G S
GS
Tanaka
N
N
D000818:Animals; D002784:Cholesterol / Q000378:metabolism; D019826:Cholic Acid / Q000096:biosynthesis*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D006868:Hydrolysis; D006900:Hydroxylation; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology / Q000378:metabolism; D011817:Rabbits; D013250:Steroid Hydroxylases / Q000378:metabolism*
D002784:Cholesterol; D013250:Steroid Hydroxylases; D019826:Cholic Acid