Cerebrospinal fluid steroidomics: are bioactive bile acids present in brain?

Abstract

In this study we have profiled the free sterol content of cerebrospinal fluid by a combination of charge tagging and liquid chromatography-tandem mass spectrometry. Surprisingly, the most abundant cholesterol metabolites were found to be C(27) and C(24) intermediates of the bile acid biosynthetic pathways with structures corresponding to 7alpha-hydroxy-3-oxocholest-4-en-26-oic acid (7.170 +/- 2.826 ng/ml, mean +/- S.D., six subjects), 3beta-hydroxycholest-5-en-26-oic acid (0.416 +/- 0.193 ng/ml), 7alpha,x-dihydroxy-3-oxocholest-4-en-26-oic acid (1.330 +/- 0.543 ng/ml), and 7alpha-hydroxy-3-oxochol-4-en-24-oic acid (0.172 +/- 0.085 ng/ml), and the C(26) sterol 7alpha-hydroxy-26-norcholest-4-ene-3,x-dione (0.204 +/- 0.083 ng/ml), where x is an oxygen atom either on the CD rings or more likely on the C-17 side chain. The ability of intermediates of the bile acid biosynthetic pathways to activate the liver X receptors (LXRs) and the farnesoid X receptor was also evaluated. The acidic cholesterol metabolites 3beta-hydroxycholest-5-en-26-oic acid and 3beta,7alpha-dihydroxycholest-5-en-26-oic acid were found to activate LXR in a luciferase assay, but the major metabolite identified in this study, i.e. 7alpha-hydroxy-3-oxocholest-4-en-26-oic acid, was not an LXR ligand. 7Alpha-hydroxy-3-oxocholest-4-en-26-oic acid is formed from 3beta,7alpha-dihydroxycholest-5-en-26-oic acid in a reaction catalyzed by 3beta-hydroxy-Delta(5)-C(27)-steroid dehydrogenase (HSD3B7), which may thus represent a deactivation pathway of LXR ligands in brain. Significantly, LXR activation has been found to reduce the symptoms of Alzheimer disease (Fan, J., Donkin, J., and Wellington C. (2009) Biofactors 35, 239-248); thus, cholesterol metabolites may play an important role in the etiology of Alzheimer disease.

SCHEME 1.

Sample preparation for analysis of sterols and bile acids by LC-MSⁿ. By analyzing samples in parallel by routes A (without cholesterol oxidase) and B (with cholesterol oxidase), analytes possessing a 3-oxo group are differentiated from those oxidized to contain one (i.e. 3β-hydroxy-5-ene or 3β-hydroxy-5α-hydrogen sterols/bile acids).

SCHEME 2.

Charge tagging of sterols and bile acids as exemplified by 3β-hydroxycholest-5-en-26-oic acid.

SCHEME 3.

Fragmentation of GP-tagged sterols and bile acids. a, major MS² fragmentation routes for GP-tagged sterols and bile acids exemplified by 27-hydroxycholesterol. A 3-oxo-4-ene functionality was generated by oxidation of the native 3β-hydroxy-5-ene function by cholesterol oxidase prior to treatment with GP reagent. b and c, structurally informative fragment ions observed in MS³ ([M]⁺→[M − 79]⁺→) spectra of GP-tagged sterols exemplified by 27-hydroxycholesterol following cholesterol oxidase treatment (b) and 7α-hydroxycholesterol after similar treatment (c).

FIGURE 1.

Identification of monohydroxycholesterols and hydroxynorcholestenedione in CSF. a, MRM chromatogram for the transition 534→455→. b, RIC for the exact m/z 534.3690 ± 5 ppm. c–g, MS³ [534→455→] spectra of the peaks eluting as follows: 4.82 min, peak 1 (c); 7.42 min, peak 3 (d); 7.57 min, peak 4 (e); 7.95 min, peak 5 (f); and 10.05 min, peak 8 (g); in chromatogram a. The MRM chromatogram (a) and MS³ spectra (c–g) were generated in the LIT, the RIC (b) was generated in parallel in the Orbitrap analyzer. The MS³ spectra correspond to GP-tagged 7α-hydroxy-26-norcholest-4-ene-3,x-dione (c); 24(S)-hydroxycholesterol (d); 25-hydroxycholesterol (e); 27-hydroxycholesterol (f); and 7α-hydroxycholesterol (g). Proposed structures of the GP-tagged molecules are shown as insets to the appropriate spectra. GP-tagged 7α-hydroxy-26-norcholest-4-ene-3,x-dione appears in chromatograms (a and b) as syn and anti conformers (peaks 1 and 2). Chromatograms and spectra are from sterols isolated from CSF. Some confusion may arise concerning the nomenclature of 27-hydroxycholesterol and related compounds. According to rules of priority of numbering the correct description of 27-hydroxycholesterol is 25(R),26-hydroxycholesterol. However, the common name is 27-hydroxycholesterol on account of its formation via the mitochondrial CYP27A1-catalyzed hydroxylation of cholesterol, and this will be the name used here, although we will use the abbreviation C⁵-3β,26-diol in accord with the systematic name cholest-5-ene-3β,26-diol recommended by Fahy et al. (74). In other cases, we adopt the nomenclature recommended by Fahy et al. (74) and the Lipid Maps consortium. %RA, % relative abundance.

FIGURE 2.

Identification of hydroxycholestenoic and hydroxyoxocholestenoic acids in CSF. a and b, RICs for the exact m/z 548.3847 (a) and 564.3796 (b) ±5 ppm. c, MS³ [548→469→] spectrum of the peak eluting at 7.63 min in RIC (a). d, MS³ [564→485→] spectrum of the peak eluting at 5.91 min in RIC (b). The MS³ spectra correspond to GP-tagged 3β-hydroxycholest-5-en-26-oic (c) and 7α-hydroxy-3-oxocholest-4-en-26-oic acids (d). Structures of the GP-tagged molecules are shown as insets to the appropriate spectra. GP-tagged 7α-hydroxy-3-oxocholest-4-en-26-oic acid appears as syn and anti conformers in RIC (b). The syn and anti confirmers appear to give split peaks in RIC (b) (i.e. 5.69 and 5.91 min and 6.47 and 6.67 min). The MS³ spectra for these peaks are indistinguishable; however, only the latter eluting peaks (5.91 min, 6.67 min) appear in the RIC of the authentic reference compound. The origin of the early eluting peaks is unknown but may be related to the stereochemistry of the C-17 side chain. 7β-Hydroxy-3-oxocholest-4-en-26-oic is the minor component eluting at 4.18 min in RIC (b). Chromatograms and spectra are from sterols isolated from CSF, and recorded as indicated in Fig. 1. %RA, % relative abundance.

FIGURE 3.

Identification of hydroxybisoxocholestenoic and dihydroxyoxocholestenoic acids. a and b, RICs for the exact m/z 578.3589 (a) and 580.3745 (b) ± 5 ppm. c, MS³ [578→499→] spectrum of the peak eluting at 2.35 min in RIC (a). d, MS³ [580→501→] spectrum of the peak eluting at 2.67 min in RIC (b). The MS³ spectra correspond to GP-tagged 7α-hydroxy-3,x-bisoxocholest-4-en-26-oic acid (c), and 7α,x-dihydroxy-3-oxocholest-4-en-26-oic acid (d). Proposed structures of the GP-tagged molecules are shown as insets to the appropriate spectra. GP-tagged 7α,x-dihydroxy-3-oxocholest-4-en-26-oic acid appears as syn and anti conformers appearing at 2.67 and 3.79 min in RIC (b). Chromatograms and spectra are from sterols isolated from CSF and recorded as indicated in Fig. 1. %RA, % relative abundance.

SCHEME 4.

Suggested pathways for biosynthesis of bile acids found in CSF. The acidic pathway starts with 26-hydroxylation of cholesterol by CYP27A1, and the 24(S)-hydroxycholesterol pathway by 24(S)-hydroxylation of cholesterol by CYP46A1. Compounds drawn in black are identified in CSF, those in blue are presumptively identified, and those in red were not detected or were present in trace quantities only. With the exception of α-methylacyl-CoA racemase (necessary for the acidic pathway), all of the enzymes (or mRNA transcripts) required for the formation of 7α-hydroxy-3-oxochol-4-en-24-oic acid are present in brain. Abbreviations, Swiss-Prot accession number, and reference to the enzyme, where applicable (or mRNA transcript), in brain are as follows: CYP27A1, cytochrome P450 27A1, Q02318 (54); CYP46A1, cytochrome P450 46A1, Q9Y6A2 (48); CYP7B1, cytochrome P450 7B1, O75881 (75); CYP39A1, cytochrome P450 39A1, Q9NYL5 (53); HSD3B7, 3β-hydroxysteroid dehydrogenase type 7, Q9H2F3 (24); VLCS, very long chain acyl-CoA synthetase, O35488 (56); AMACR, α-methylacyl-CoA racemase, Q9UHK6; BCOX, branched chain acyl-CoA oxidase, Q99424 (76); DBP, d-bifunctional protein, P51659 (57); LBP, l-bifunctional protein, Q08426 (57); SCPx, sterol carrier protein x, P22307 (77). Metabolites identified as LXRα ligands in SN4741 cells are indicated by LXR+.

FIGURE 4.

Identification of C₂₄ bile acids in CSF. RICs for the exact m/z 522.3326 (a) and 540.3432 (b) ± 5 ppm are shown. c, MS³ [522→443→] spectrum of the peak eluting at 2.08 min in RIC (a). d, MS³ [540→461→] spectrum of the peak eluting at 1.79 min in RIC (b). The MS³ spectra correspond to GP-tagged 7α-hydroxy-3-oxochol-4-en-24-oic (c) and a trihydroxycholanoic acid, possibly 3β,x,y-trihydroxy-5α-cholan-24-oic acid (d). Proposed structures of the GP-tagged molecules are shown as insets to the appropriate spectra. Chromatograms and spectra are from sterols isolated from CSF and recorded as indicated in Fig. 1. %RA, % relative abundance.

FIGURE 5.

Analysis of the nuclear receptor activational capacity of acidic intermediates of bile acid biosynthesis. Analysis of luciferase activity in SN4741 cells transfected with an LXR-responsive luciferase reporter construct (LXRE) and LXRα, as indicated, and stimulated for 24 h with 22(R)-hydroxycholesterol (cholest-5-ene-3β,22(R)-diol, C⁵-3β,22R-diol; 10 μm) a known LXRα ligand (62, 63), or the acidic compounds indicated, i.e. 3β-hydroxycholest-5-en-26-oic acid, CA⁵-3β-ol; 3β,7α-dihydroxycholest-5-en-26-oic acid, CA⁵-3β,7α-diol; 7α,12α-dihydroxy-3-oxo-5β-cholan-24-oic acid, 5β-BA-7α,12α-diol-3-one; 7α-hydroxy-3-oxocholest-4-en-26-oic acid, CA⁴-7α-ol-3-one; and 3-oxo-5β-cholan-24-oic acid, 5β-BA-3-one. The firefly luciferase activity was normalized to Renilla luciferase activity, and the values are expressed as fold activation over the normalized basal LXR response element-luciferase activity set to 1. Data are means ± S.E. (n = 3), *, p < 0.05; **, p < 0.01 compared with vehicle treatment. %RA, % relative abundance.