Cholesterolomics: An update

Abstract

Cholesterolomics can be regarded as the identification and quantification of cholesterol, its precursors post squalene, and metabolites of cholesterol and of its precursors, in a biological sample. These molecules include 1,25-dihydroxyvitamin D₃, steroid hormones and bile acids and intermediates in their respective biosynthetic pathways. In this short article we will concentrate our attention on intermediates in bile acid biosynthesis pathways, in particular oxysterols and cholestenoic acids. These molecular classes are implicated in the aetiology of a diverse array of diseases including autoimmune disease, Parkinson's disease, motor neuron disease, breast cancer, the lysosomal storage disease Niemann-Pick type C and the autosomal recessive disorder Smith-Lemli-Opitz syndrome. Mass spectrometry (MS) is the dominant technology for sterol analysis including both gas-chromatography (GC)-MS and liquid chromatography (LC)-MS and more recently matrix-assisted laser desorption/ionisation (MALDI)-MS for tissue imaging studies. Here we will discuss exciting biological findings and recent analytical improvements.

Keywords: Cholestenoic acids; Cholesterol; Derivatisation; Mass spectrometry; Oxysterols.

Fig. 1

Synthesis and metabolism of repressors of SREBP processing and LXR ligands, 24S,25-EC, 25-HC, of the EBI2 ligand 7α,25-diHC and of the RORγt ligand 7β,26-diHC. Shown in the inset is 24S-HC, a repressor of SREBP processing and an LXR ligand formed from cholesterol by the enzyme CYP46A1. Full stereochemistry is shown only for the initial sterols in the pathways. Systematic names given in parenthesis. Enzymes are indicated where known. A broken arrows indicates a postulated pathway with associated enzyme.

Fig. 2

Early steps of the “acidic” bile acid biosynthesis pathway generating 3β-HCA and 3β,7α-diHCA, two cholestenoic acids regulating the survival and death of motor neurons. Full stereochemistry is shown only for the initial sterol in the pathway. Systematic names given in parenthesis. Enzymes are indicated where known.

Fig. 3

Synthesis and metabolism of oxysterol modulators of Hh signalling. Some of the oxysterols are prevalent in patients suffering from NP-C disease or SLOS. Full stereochemistry is shown only for the initial sterols in the pathways. Systematic names given in parenthesis. Enzymes are indicated where known. Broken arrows indicate postulated pathway and enzyme.

Fig. 4

Oxysterols implicated in the cause of cancer and metabolism of 5,6-EC. Full stereochemistry is shown only for the initial sterol in the pathways. Systematic names given in parenthesis. Enzymes are indicated where known. Broken arrows indicate postulated pathway.

Fig. 5

Some primary products of 7-DHC oxidation found in patients with SLOS. Further metabolic products are shown in Fig. 3. Full stereochemistry is shown only for the initial sterol in the pathways. Systematic names given in parenthesis. Enzymes are indicated where known. Broken arrows indicate postulated pathway.

Fig. 6

Derivatisation reactions for sterol and oxysterol analysis by LC-MS (A) Derivatisation with PTAD. (B) Derivatisation with GP reagent. (C) Left hand side, derivatisation with dimethylglycine; right hand side, derivatisation with nicotinic acid. Abbreviations: DMAP, 4-(dimethylamino)pyridine; DMG, N,N-dimethylglycine; EDC, 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide; DPC, N,N-diisopropylcarbodiimide.