Analysis by liquid chromatography-mass spectrometry of sterols and oxysterols in brain of the newborn Dhcr7(Δ3-5/T93M) mouse: a model of Smith-Lemli-Opitz syndrome

Abstract

In this study the sterol and oxysterol profile of newborn brain from the Dhcr7(Δ3-5/T93M) mouse model of Smith-Lemli-Opitz syndrome (SLOS) has been investigated. This is a viable mouse model which is compound heterozygous containing one null allele and one T93M mutation on Dhcr7. We find the SLOS mouse has reduced levels of cholesterol and desmosterol and increased levels of 7- and 8-dehydrocholesterol and of 7- and 8-dehydrodesmosterol in brain compared to the wild type. The profile of enzymatically formed oxysterols in the SLOS mouse resembles that in the wild type but the level of 24S-hydroxycholesterol, the dominating cholesterol metabolite, is reduced in a similar proportion to that of cholesterol. A number of oxysterols abundant in the SLOS mouse are probably derived from 7-dehydrocholesterol, however, the mechanism of their formation is unclear.

Fig. 1

Charge-tagging of sterols/oxysterols. (A) Oxidation of 3β-hydroxy-5-ene steroids to 3-oxo-4-ene equivalents followed by derivatisation with GP reagent. (B) MS² fragmentation of GP-tagged steroids. (C) MS³ fragmentation of the [M−79]⁺ ion from B. In A, B and C, 24S-hydroxycholesterol is the exemplar. (D) The effect of 7-hydroxylation on MS³ fragmentation. (E) MS³ fragmentation of GP-tagged 7-DHC. (F) MS³ fragmentation of GP-tagged 8-DHC.

Fig. 2

LC–MS(MS)ⁿ analysis of DHCs and DHDs from newborn Dhcr7^Δ3-5/T93M mouse brain following GP charge-tagging. (A) Upper panel, LC–MS RIC for m/z 516.3948 ± 10 ppm corresponding to DHCs. Lower panel, LC–MS RIC for m/z 514.3792 corresponding to DHDs. The short gradient was employed. MS³ (516 → 437→) spectra of the peaks eluting at (B) 11.08 min corresponding to desmosterol (cholesta-5,24-dien-3β-ol); (C) 11.71 min corresponding to 7-DHC (cholesta-5,7-dien-3β-ol); (D) authentic 7-DHC; (E) 11.91 min corresponding to 8-DHC (cholesta-5,8(9)-dien-3β-ol); (F) authentic 8-DHC. MS³ (514 → 435→) spectra of peaks eluting at (G) 10.61 min corresponding to 7-DHD (cholesta-5,7,24-trien-3β-ol) and (H) 11.29 min corresponding to 8-DHD (cholesta-5,8(9),24-trien-3β-ol). Authentic standards were not available for 7- or 8-DHD, hence identifications are presumptive. Shown as insets in each spectrum are the structures of the GP-tagged molecules.

Fig. 3

LC–MS(MS)ⁿ analysis of monohydroxycholesterols from newborn Dhcr7^Δ3-5/T93M mouse brain following GP charge-tagging. (A) LC-MS RIC for m/z 534.4054 ± 10 ppm using the short gradient. Shown in the inset is the chromatographic separation of 24S-, 25-, 24R and 26-hydroxycholesterols using the longer gradient. MS³ (534 → 455→) spectra of peaks eluting at (B) 6.06 min (short gradient) corresponding to 22R-hydroxycholesterol (cholest-5-ene-3β,22R-diol); (C) 7.36 min (short gradient, 16.22 min longer gradient) corresponding to 24S-hydroxycholesterol (cholest-5-ene-3β,24S-diol); (D) 17.41 min (longer gradient) corresponding to 25-hydroxycholesterol (cholest-5-ene-3β,25-diol); (E) 19.22 min (longer gradient, 7.84 min short gradient) corresponding to 24R-hydroxycholesterol (cholest-5-ene-3β,24R-diol); (F) 19.59 min (longer gradient) corresponding to (25R)26-hydroxycholesterol (cholest-(25R)-5-ene-3β,26-diol); (G) 9.59 min (short gradient) corresponding to 7β-hydroxycholesterol (cholest-5-ene-3β,7β-diol) and (H) 10.16 min (short gradient) corresponding to 7α-hydroxycholesterol (cholest-5-ene-3β,7α-diol). Other peaks eluting with the short gradient displayed in chromatogram 3A correspond to the second conformers of 24S-hydroxycholesterol (7.67 min) and of 7β-hydroxycholesterol (10.00 min) and to 6β-hydroxycholesterol (10.51 min). The peak at 18.03 min eluting with the longer gradient corresponds to the second conformer of 24S-hydroxycholesterol. Shown as insets in each spectrum are the structures of the GP-tagged molecules.

Fig. 4

LC–MS(MS)ⁿ analysis for 24S,25-epoxycholesterol and other oxysterols of similar m/z from newborn Dhcr7^Δ3-5/T93M mouse brain following GP charge-tagging. The short gradient was employed throughout. (A) LC-MS RIC of m/z 532.3898 ± 10 ppm. MS³ (532 → 453→) spectra of peaks eluting at (B) 6.68 min corresponding to 24S,25-epoxycholesterol (3β-hydroxycholest-5-en-24S,25-epoxide) and (C) 7.72 min corresponding to 24-oxocholesterol (3β-hydroxycholest-5-en-24-one). (D) Upper panel, LC–MS RIC of m/z 550.4003 ± 10 ppm corresponding to dihydroxycholesterols. Lower panel, LC–MS RIC of m/z 564.4160 ± 10 ppm corresponding to hydroxymethoxycholesterol. (E) MS³ (550 → 471→) spectrum of 24,25-dihydroxycholesterol (cholest-5-ene-3β,24,25-triol) eluting at 3.84 min. (F) MS³ spectrum of 24-hydroxy-25-methoxycholesterol (3β,24-dihydroxycholest-5-ene-25-methoxide) eluting at 6.06 min (the spectrum could also correspond to the 25-hydroxy-24-methoxy isomer). 24-Oxocholesterol, 24,25-dihydroxycholesterol and 24-hydroxy-25-methoxycholesterol (and/or its 25-hydroxy-24-methoxy isomer) are all formed from 24S,25-epoxycholesterol during GP-derivatisation. Shown as insets in each spectrum are the structures of the GP-tagged molecules.

Fig. 5

MS³ (532→453→) spectra of other GP-tagged oxysterols eluting in chromatogram 4A. (A) (24Z)26-Hydroxydesmosterol (cholesta-5,24(Z)-diene-3β,26-diol) eluting at 7.10; (B) 23-hydroxydesmosterol (cholesta-5,24-diene-3β,23-diol) eluting at 7.36 min; (C) 22-oxocholesterol (3β-hydroxycholest-5-en-22-one) eluting at 7.98 min; (D) 7β-hydroxydesmosterol (cholesta-5,24-diene-3β,7β-diol) eluting at 8.60 min and (E) 7α-hydroxydesmosterol (cholesta-5,24-diene-3β,7α-diol) eluting at 9.33 min. Authentic standards are only available for (24Z)26-hydroxydesmosterol and 22-oxocholesterol. All other oxysterols are presumptively identified. Shown as insets in each spectrum are the structures of the GP-tagged molecules.

Fig. 6

Oxysterols derived from 7-DHC or cholesterol possibly via free radical oxidation/autoxidation. MS³ spectra (532 → 453→) of GP-tagged oxysterols eluting in chromatogram 4A at (A) 9.64 min and (B) 10.10 min possibly corresponding to 4β-hydroxy-7-dehydrocholesterol (cholesta-5,7-diene-3β,4β-diol) and its 4α isomer, respectively; and (C) 10.45 min corresponding to cholest-4-ene-3,6-dione. (D) RIC of m/z 548.3847 ± 10 ppm. MS³ (548 → 469→) spectra of GP-tagged oxysterols eluting at (E) 8.34 min and (F) 9.07 min possibly both corresponding to 3β,5α-dihydroxycholest-7-en-6-one and (G) 10.10 min probably corresponding to an additional dihydroxycholestenone isomer. Authentic standards are not available for 4-hydroxy-7-dehydrocholesterol or dihydroxycholestenone isomers hence their identifications are presumptive. Shown as insets in each spectrum are the structures of the GP-tagged molecules.

Fig. 7

Levels of oxysterols in brain of newborn Dhcr7^Δ3-5/T93M and phenotypically normal Dhcr7^+/T93M control mice. Data for Dhcr7^Δ3-5/T93M mice (n = 4) is in red and that for Dhcr7^+/T93 control mice (n = 6) in blue. Statistical analysis was performed by Student’s t-test, P < 0.05 was considered a statistically significant difference (*), P < 0.01 (**), P < 0.001 (***). Data represent mean ± SE.