Identification of 7α,24-dihydroxy-3-oxocholest-4-en-26-oic and 7α,25-dihydroxy-3-oxocholest-4-en-26-oic acids in human cerebrospinal fluid and plasma

Abstract

Dihydroxyoxocholestenoic acids are intermediates in bile acid biosynthesis. Here, using liquid chromatography - mass spectrometry, we confirm the identification of 7α,24-dihydroxy-3-oxocholest-4-en-26-oic and 7α,25-dihydroxy-3-oxocholest-4-en-26-oic acids in cerebrospinal fluid (CSF) based on comparisons to authentic standards and of 7α,12α-dihydroxy-3-oxocholest-4-en-26-oic and 7α,x-dihydroxy-3-oxocholest-4-en-26-oic (where hydroxylation is likely on C-22 or C-23) based on exact mass measurement and multistage fragmentation. Surprisingly, patients suffering from the inborn error of metabolism cerebrotendinous xanthomatosis, where the enzyme CYP27A1, which normally introduces the (25 R)26-carboxylic acid group to the sterol side-chain, is defective still synthesise 7α,24-dihydroxy-3-oxocholest-4-en-26-oic acid and also both 25 R- and 25 S-epimers of 7α,12α-dihydroxy-3-oxocholest-4-en-26-oic acid. We speculate that the enzymes CYP46A1 and CYP3A4 may have C-26 carboxylase activity to generate these acids. In patients suffering from hereditary spastic paraplegia type 5 the CSF concentrations of the 7α,24- and 7α,25-dihydroxy acids are reduced, suggesting an involvement of CYP7B1 in their biosynthesis in brain.

Keywords: Bile acid; Brain; Cerebrotendinous xanthomatosis; Cytochrome P450; Hereditary spastic paraplegia type 5; Oxysterol.

Graphical abstract

Fig. 1

Intermediates in bile acid biosynthesis pathways. (A) Formation of dihydroxy-3-oxocholest-4-en-26-oic acids from cholesterol via the acidic, neutral and 25-hydroxylase pathways [1,2]. In the acidic and neutral pathway stereochemistry at C-25 is assumed to be 25 R unless otherwise indicated. (B) Formation of (25 R)CA⁴-7α,24 S-diol-3-one and (25 S)CA⁴-7α,24 S-diol-3-one via the 24 S-hydroxylase pathway (red box) [21] and of 25 S- and 25 R-epimers of CA⁴-7α,12α-diol-3-one in the absence of CYP27A1 [26] (gold box). For simplicity the formation and hydrolysis of CoA-thioesters in the steps preceding and succeeding C-25 racemisation are not shown. (C) Latter steps of the acidic pathway proceeding mostly in the peroxisome leading to the four diastereomers of CA⁴-7α,24-diol-3-one. In liver the dominant product is (25 R)CA⁴-7α,24 R-diol-3-one. The inset shows the formation of (25 S)CA⁴-7α,24 R-diol-3-one from its CoA-thioester following C-25 racemisation. In CTX the enzyme CYP27A1 is deficient, while in SPG5 CYP7B1 is deficient. Abbreviations: ACOX2: acyl-coenzyme A oxidase 2. AMACR: α-methylacyl-CoA racemase. BACS: bile acid-CoA synthetase. C: Cholestane. CA: Cholestanoic acid. CH25H: Cholesterol 25-hydroxylase. CYP: Cytochrome P450. DBP: D-Bifunctional Protein. HSD3B7: 3β-Hydroxysteroid dehydrogenase type 7. LBP: L-Bifunctional protein. SPCx: Sterol carrier protein x. VLCS: Very long chain Co-A synthetase. The dihydroxy-3-oxocholest-4-en-26-oic acids CA⁴-7α,25-diol-3-one, CA⁴-7α,12α-diol-3-one and CA⁴-7α,24-diol-3-one are colour coded green, gold and red, respectively.

Fig. 2

Conversion of 3β-hydroxy-5-ene acids to their 3-oxo-4-ene analogues by cholesterol oxidase enzyme from Streptomyces Sp and derivatisation with [²H₅]GP to give syn and anti conformers. In CA⁵-3β,7α,24 S-triol and CA⁴-7α,24 S-diol-3-one R₁ = OH and R₂ = H. In CA⁵-3β,7α,25-triol and CA⁴-7α,25-diol-3-one R₁ = H and R₂ = OH (upper panel). Structures of (25 S)CA⁴-7α,25-diol-3-one and its isomers (25 R)CA⁴-7α,25-diol-3-one, (25 R)CA⁴-7α,24-diol-3-one and (25 R)CA⁴-7α,12α-diol-3-one are shown in the lower panel.

Fig. 3

LC-HRMS RICs (585.4059 ± 10 ppm) corresponding to [M]⁺ ions of [²H₅]GP derivatised dihydroxy-3-oxocholest-4-en-26-oic acids. (A) Control CSF (upper panel), authentic standard of (25 R)CA⁴-7α,24 S-diol-3-one (lower panel). (B) Control CSF (upper panel), authentic standard of (25 R/S)CA⁴-7α,25-diol-3-one (lower panel). (C) Control CSF (SPE1-Fr1A) treated with cholesterol oxidase followed by [²H₅]GP reagent (upper panel), control CSF (SPE1-Fr1B) treated with [²H₀]GP reagent in the absence of cholesterol oxidase (lower panel). In (C) the RICs for [²H₅]GP derivatised and [²H₀]GP derivatised acids are plotted with the same y-axis. Data was acquired in the Orbitrap analyser of the Orbitrap Elite instrument. Peaks corresponding to the dihydroxy-3-oxocholest-4-en-26-oic acids CA⁴-7α,25-diol-3-one, CA⁴-7α,12α-diol-3-one, CA⁴-7α,24-diol-3-one and CA⁴-7α,x-diol-3-one are colour coded green, gold, red and purple respectively.

Fig. 4

MS³ ([M]⁺→[M-Py]⁺→, i.e. 585.4→501.3→) spectra acquired in the analyses of control CSF (A, C, E & F) and authentic standards of (25 R)CA⁴-7α,24 S-diol-3-one (B) and (25 R/S)CA⁴-7α,25-diol-3-one (D). The spectra confirm the identification of CA⁴-7α,24-diol-3-one in (A), of CA⁴-7α,25-diol-3-one in (C), of CA⁴-7α,x-diol-3-one in (E) and of CA⁴-7α,12α-diol-3-one in (F). Data was acquired in the linear ion trap (LIT) analyser of the Orbitrap Elite hybrid instrument with an accuracy of m/z ± 0.1 for most fragment-ions.

Fig. 5

LC-MS³ MRM transitions appropriate to (A) CA⁴-7α,24-diol-3-one, (B) CA⁴-7α,25-diol-3-one, (C) CA⁴-7α,x-diol-3-one and (D) CA⁴-7α,12α-diol-3-one from the analysis of a control CSF sample. MRMs were generated from data acquired in the LIT analyser of the Orbitrap Elite hybrid instrument with an m/z window of ±0.4. Peaks are colour coded as in Fig. 3.

Fig. 6

Analysis of CSF from a CTX patient. (A) LC-HRMS RICs (585.4059 ± 10 ppm, upper panel; 580.3745 ± 10 ppm, lower panel). The upper panel shows CTX CSF (SPE1-Fr1A) treated with cholesterol oxidase followed by [²H₅]GP reagent, while the lower panel shows CTX CSF (SPE1-Fr1B) treated with [²H₀]GP reagent in the absence of cholesterol oxidase. The RICs for [²H₅]GP derivatised and [²H₀]GP derivatised acids are plotted with the same y-axis. Data was acquired in the Orbitrap analyser. (B) LC-MS³ (585.4→501.3→) TIC appropriate to [²H₅]GP derivatised dihydroxy-3-oxocholest-4-en-26-oic acids. LC-MS³ MRM transitions appropriate to (C) CA⁴-7α,24-diol-3-one, (D) CA⁴-7α,25-diol-3-one, (E) CA⁴-7α,x-diol-3-one and (F) CA⁴-7α,12α-diol-3-one. In (D) and (E) the horizontal bars indicate where the targeted acids are expected to elute. The MRMs in (C–F) were generated from data acquired in the LIT analyser of the Orbitrap Elite hybrid instrument with an m/z window of ±0.4. Chromatograms displayed in Fig. 5, Fig. 6 were recorded on different days and show a time shift of about 0.4 min for the later eluting peaks. Retention times were correlated by the analysis of QC samples within in each sample batch. Peaks are colour coded as in Fig. 3.

Fig. 7

MS³ ([M]⁺→[M-Py]⁺→, i.e. 585.4→501.3→) spectra from CSF of a CTX patient. The spectra confirm the presence of dihydroxy-3-oxocholest-4-en-26-oic acids in CSF. Spectra shown in (A) & (B) are of syn and anti conformers of CA⁴-7α,24-diol-3-one, (C) & (E) are interpreted as syn and anti conformers of (25 S)CA⁴-7α,12α-diol-3-one, and (D) & (F) are interpreted as syn and anti conformers of (25 R)CA⁴-7α,12α-diol-3-one. Data was acquired in the LIT analyser of the Orbitrap Elite hybrid instrument with an accuracy of m/z ± 0.1 for most fragment-ions.

Fig. 8

LC-HRMS RICs ±10 ppm appropriate to the analysis of [²H₅]GP and [²H₀]GP derivatised dihydroxy-3-oxocholest-4-en-26-oic acids in control (A), CTX (C) and SPG5 (D) plasma. The RICs for [²H₅]GP derivatised dihydroxy-3-oxocholest-4-en-26-oic acids are plotted on the same y-axis scale as the RICs for [²H₀]GP derivatised acids. (B) LC-MS³ TICs appropriate to [²H₅]GP and [²H₀]GP derivatised dihydroxy-3-oxocholest-4-en-26-oic acids in control plasma. Control, CTX and SPG5 plasma were analysed on different days which causes a small deviation in retention time. Peaks were correlated by recording QCs with each sample batch. HRMS data was recorded in the Orbitrap and MS³ data in the LIT of the Orbitrap Elite hybrid instrument. Peaks are colour coded as in Fig. 3.