Deep mining of oxysterols and cholestenoic acids in human plasma and cerebrospinal fluid: Quantification using isotope dilution mass spectrometry

Abstract

Both plasma and cerebrospinal fluid (CSF) are rich in cholesterol and its metabolites. Here we describe in detail a methodology for the identification and quantification of multiple sterols including oxysterols and sterol-acids found in these fluids. The method is translatable to any laboratory with access to liquid chromatography – tandem mass spectrometry. The method exploits isotope-dilution mass spectrometry for absolute quantification of target metabolites. The method is applicable for semi-quantification of other sterols for which isotope labelled surrogates are not available and approximate quantification of partially identified sterols. Values are reported for non-esterified sterols in the absence of saponification and total sterols following saponification. In this way absolute quantification data is reported for 17 sterols in the NIST SRM 1950 plasma along with semi-quantitative data for 8 additional sterols and approximate quantification for one further sterol. In a pooled (CSF) sample used for internal quality control, absolute quantification was performed on 10 sterols, semi-quantification on 9 sterols and approximate quantification on a further three partially identified sterols. The value of the method is illustrated by confirming the sterol phenotype of a patient suffering from ACOX2 deficiency, a rare disorder of bile acid biosynthesis, and in a plasma sample from a patient suffering from cerebrotendinous xanthomatosis, where cholesterol 27-hydroxylase is deficient.

Cholesterol metabolism: from lipidomics to immunology

“…Cholesterol accumulation in the interior of the compartment was reasoned to mediate the anti-viral activity of both 25-HC and ICZ (14). A complication to this mechanism is that in cellular assays 25-HC travels directly to the endoplasmic reticulum without traversing the lysosome on its way to inhibiting SREBP-2 processing (34,112), however, in vivo it is likely that when derived from the circulation, where the majority of 25-HC is esterified (113,114), the ester as part of lipoprotein particles is taken-up by receptor mediated endocytosis and travels to the lysosome where it is hydrolysed to the non-esterified molecule. Zang et al concluded that 25-HC reduces SARS-CoV-2 spike-mediated fusion via a mechanism which involves altered cholesterol levels leading to inhibition of viral replication (14) (115,116).…”

“…Interestingly, these results were not replicated in a mouse model of NASH where levels of 7α,26-diHC and 7α,26-diHCO were about 25 ng/g and 50 ng/g, respectively, in both WT and treated animals (131). It is important to note that both 7α,26-diHC and 7α,26-diHCO are found in man and mouse as 25Rand 25S-epimers where the stereochemistry at C-25 is different (113,132), and that Raselli specifically quantified the 25R-epimer (131). Very few other studies define the specific epimer(s) measured, however, the 25R-epimers is usually dominant except in the case of CYP27A1 deficiency (113,132,133), where the 25S-epimer dominates.…”

“…It is important to note that both 7α,26-diHC and 7α,26-diHCO are found in man and mouse as 25Rand 25S-epimers where the stereochemistry at C-25 is different (113,132), and that Raselli specifically quantified the 25R-epimer (131). Very few other studies define the specific epimer(s) measured, however, the 25R-epimers is usually dominant except in the case of CYP27A1 deficiency (113,132,133), where the 25S-epimer dominates. By default, and unless explicitly stated here, it can be assumed that the stereochemistry at C-25 is R and the concentrations presented are for the 25R-epimer or for a combination of C-25R and C-25S epimers.…”

“…Both 7α,25-diHC (1 ng/mL ) and 7α,26-diHC (6 -10 ng/mL) can be detected by LC-MS in human plasma following ester hydrolysis as the sum of esterified and the non-esterified sterols (113,134), but their levels as free non-esterified molecules are very low (7α,25-diHC, 0.2 ng/mL; 7α,26-diHC, 0.5 ng/mL, ( 135)). However, the non-esterified 3-oxo-4-ene metabolites (produced through the action of HSD3B7 are more abundant (7α,25-diHCO, 1 -2 ng/mL; 7α,26-diHCO, 3 -8 ng/mL (113,135)). In mouse plasma, the situation is similar with levels of non-esterified 7α,25-diHC and 7α,26-diHC being very low at about 0.1 ng/mL, with 7α,25-diHCO and 7α,26-diHCO at about 1 ng/mL (132).…”

Progress and Challenges in Quantifying Carbonyl-Metabolomic Phenomes with LC-MS/MS

“…Since GP can label sterols with a carbonyl group, the sterols with a hydroxyl group and oxysterols with a carbonyl group cannot be analyzed in a single LC-MS analysis using GP derivatization. However, GP/d 5 -GP can be used to quantify oxysterols and sterols simultaneously if sterols are oxidized by cholesterol oxidase (COD) first to contain a carbonyl group (Scheme 3) [137]. On the basis of this principle, it is possible to quantify up to four different samples in a single run by using differential mass tags and isobaric mass tags simultaneously through multiplex methods [138].…”