Abstract
Cholestane-3β,5α,6β-triol (3β,5α,6β-triol) is formed from cholestan-5,6-epoxide (5,6-EC) in a reaction catalysed by cholesterol epoxide hydrolase, following formation of 5,6-EC through free radical oxidation of cholesterol. 7-Oxocholesterol (7-OC) and 7β-hydroxycholesterol (7β-HC) can also be formed by free radical oxidation of cholesterol. Here we investigate how 3β,5α,6β-triol, 7-OC and 7β-HC are metabolised to bile acids. We show, by monitoring oxysterol metabolites in plasma samples rich in 3β,5α,6β-triol, 7-OC and 7β-HC, that these three oxysterols fall into novel branches of the acidic pathway of bile acid biosynthesis becoming (25R)26-hydroxylated then carboxylated, 24-hydroxylated and side-chain shortened to give the final products 3β,5α,6β-trihydroxycholanoic, 3β-hydroxy-7-oxochol-5-enoic and 3β,7β-dihydroxychol-5-enoic acids, respectively. The intermediates in these pathways may be causative of some phenotypical features of, and/or have diagnostic value for, the lysosomal storage diseases, Niemann Pick types C and B and lysosomal acid lipase deficiency. Free radical derived oxysterols are metabolised in human to unusual bile acids via novel branches of the acidic pathway, intermediates in these pathways are observed in plasma.
Highlights
The oxysterols 7-oxocholesterol (7-OC, for systematic names see Supplemental Table S1), 7β-hydroxycholesterol (7β-HC), 5α,6-epoxycholesterol (5α,6-EC), 5β,6-epoxycholesterol (5β,6-EC) and cholestane-3β,5α,6β-triol (3β,5α,6β-triol) have for many years been thought of as autoxidation artefacts formed ex vivo from cholesterol during sample handling [1,2,3]. This view is changing with the realisation that (i) 7-OC can be formed enzymatically from 7-dehydrocholesterol (7-DHC) [4], (ii) 7β-HC and 7-OC can be interconverted in enzyme catalysed reactions [5,6,7,8,9], while (iii) the 5α,6-EC adduct dendrogenin A (DDA) is present in tissue [10], (iv) 5,6-EC is hydrolysed by the enzyme cholesterol epoxide hydrolase (ChEH) to 3β,5α,6β-triol which itself is oxidised by hydroxysteroid dehydrogenase (HSD) 11B2 to 3β,5α-dihydroxycholestan-6-one (3β,5α-diHC-6O, called 6-oxocholestan3β,5α-diol) found at elevated levels in breast cancer tissue [11], and (v) high levels of 7-OC and 3β,5α,6β-triol are characteristic of the lysosomal storage diseases Niemann-Pick types A, B, C1 and C2 (NPA, NPB, NPC) and lysosomal acid lipase deficiency (LALD) [12,13,14,15,16,17,18]
A charge-tagging method was adopted [31,32], where sterols, including oxysterols and bile acids, with a 3β-hydroxy group were oxidised with bacterial cholesterol oxidase (ChOx) to 3-oxo analogues and derivatised with [2H5]-labelled Girard P (GP) reagent (Fig. 2), analysed by liquid chromatography – mass spectrometry (LC-MS) at high mass-resolution (120,000 at m/ z 400, full-width at half-maximum height definition) with parallel multistage fragmentation (MSn)
GP-derivatised sterols containing a 3β,5α,6β-triol function give characteristic MS3 spectra this allows their presumptive identification by LC-MS(MSn) in the absence of authentic standards
Summary
The oxysterols 7-oxocholesterol (7-OC, for systematic names see Supplemental Table S1), 7β-hydroxycholesterol (7β-HC), 5α,6-epoxycholesterol (5α,6-EC), 5β,6-epoxycholesterol (5β,6-EC) and cholestane-3β,5α,6β-triol (3β,5α,6β-triol) have for many years been thought of as autoxidation artefacts formed ex vivo from cholesterol during sample handling [1,2,3] This view is changing with the realisation that (i) 7-OC can be formed enzymatically from 7-dehydrocholesterol (7-DHC) [4], (ii) 7β-HC and 7-OC can be interconverted in enzyme catalysed reactions [5,6,7,8,9], while (iii) the 5α,6-EC adduct dendrogenin A (DDA) is present in tissue [10], (iv) 5,6-EC is hydrolysed by the enzyme cholesterol epoxide hydrolase (ChEH) to 3β,5α,6β-triol which itself is oxidised by hydroxysteroid dehydrogenase (HSD) 11B2 to 3β,5α-dihydroxycholestan-6-one (3β,5α-diHC-6O, called 6-oxocholestan3β,5α-diol) found at elevated levels in breast cancer tissue [11], and (v) high levels of 7-OC and 3β,5α,6β-triol are characteristic of the lysosomal storage diseases Niemann-Pick types A, B, C1 and C2 (NPA, NPB, NPC) and lysosomal acid lipase deficiency (LALD) [12,13,14,15,16,17,18].
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