Analysis of bioactive oxysterols in newborn mouse brain by LC/MS

Anna Meljon,Spyridon Theofilopoulos,Cedric H.L Shackleton,Gordon L Watson,Norman B Javitt,Hans-Joachim Knölker,Ratni Saini,Ernest Arenas,Yuqin Wang,William J Griffiths

doi:10.1194/jlr.d028233

Anna Meljon, Spyridon Theofilopoulos + Show 8 more

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https://doi.org/10.1194/jlr.d028233

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Abstract

Unesterified cholesterol is a major component of plasma membranes. In the brain of the adult, it is mostly found in myelin sheaths, where it plays a major architectural role. In the newborn mouse, little myelination of neurons has occurred, and much of this sterol comprises a metabolically active pool. In the current study, we have accessed this metabolically active pool and, using LC/MS, have identified cholesterol precursors and metabolites. Although desmosterol and 24S-hydroxycholesterol represent the major precursor and metabolite, respectively, other steroids, including the oxysterols 22-oxocholesterol, 22R-hydroxycholesterol, 20R,22R-dihydroxycholesterol, and the C(21)-neurosteroid progesterone, were identified. 24S,25-epoxycholesterol formed in parallel to cholesterol was also found to be a major sterol in newborn brain. Like 24S- and 22R-hydroxycholesterols, and also desmosterol, 24S,25-epoxycholesterol is a ligand to the liver X receptors, which are expressed in brain. The desmosterol metabolites (24Z),26-, (24E),26-, and 7α-hydroxydesmosterol were identified in brain for the first time.

Highlights

Unesterified cholesterol is a major component of plasma membranes
And as performed here, oxysterols are separated from cholesterol at the earliest possible stage of the sample preparation procedure
During the sample preparation procedure, an oxysterolrich fraction (SPE1-FR1) was separated from a cholesterolrich fraction (SPE1-FR3), and both fractions were analyzed in separate LC-multistage fragmentation (MSn) analysis

Summary

Introduction

Unesterified cholesterol is a major component of plasma membranes. In the brain of the adult, it is mostly found in myelin sheaths, where it plays a major architectural role. 24S,25-epoxycholesterol formed in parallel to cholesterol was found to be a major sterol in newborn brain. Cholesterol in brain can be found in three distinct pools: i) in the myelin sheaths of oligodendroglia, ii) in plasma membranes of neurons and other glial cells, and iii) in a metabolically active pool made up of other organelles, e.g., endoplasmic reticulum (ER), Golgi [2, 3]. In the newborn mouse, which is born nearly helpless, there is essentially no myelination in the brain, and cholesterol levels in brain are similar to most other organs (2–4 ␮g/mg). The newborn mouse offers an opportunity to study the sterol content of brain in the absence of masking levels of myelin-derived cholesterol. Contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health or other granting agencies

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