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Mass spectrometry imaging (MSI) is a new technique in the toolbox of the analytical biochemist. It allows the generation of a compound-specific image from a tissue slice where a measure of compound abundance is given pixel by pixel, usually displayed on a color scale. As mass spectra are recorded at each pixel, the data can be interrogated to generate images of multiple different compounds all in the same experiment. Mass spectrometry (MS) requires the ionization of analytes, but cholesterol and other neutral sterols tend to be poorly ionized by the techniques employed in most MSI experiments, so despite their high abundance in mammalian tissues, cholesterol is poorly represented in the MSI literature. In this chapter, we discuss some of the MSI studies where cholesterol has been imaged and introduce newer methods for its analysis by MSI. Disturbed cholesterol metabolism is linked to many disorders, and the potential of MSI to study cholesterol, its precursors, and its metabolites in animal models and from human biopsies will be discussed., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Dysregulated cholesterol metabolism has been linked to neurodegeneration. We previously found that free, non-esterified, 7α,(25 R )26-dihydroxycholesterol (7α,26-diHC), was significantly elevated in the cerebrospinal fluid of patients with Parkinson's disease (PD). In this study we investigated the role of 7α,26-diHC in midbrain dopamine (mDA) neuron development and survival. We report that 7α,26-diHC induces apoptosis and reduces the number of mDA neurons in hESC-derived cultures and in mouse progenitor cultures. Voriconazole, an oxysterol 7α-hydroxylase (CYP7B1) inhibitor, increases the number of mDA neurons and prevents the loss of mDA neurons induced by 7α,26-diHC. These effects are specific since neither 7α,26-diHC nor voriconazole alter the number of Islet1 + oculomotor neurons. Furthermore, our results suggest that elevated 24( S ),25-epoxycholesterol, which has been shown to promote mDA neurogenesis, may be partially responsible for the effect of voriconazole on mDA neurons. These findings suggest that voriconazole, and/or other azole CYP7B1 inhibitors may have implications in PD therapy development., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: WJG and YW are listed as inventors on the patent “Kit and method for the quantitative detection of steroids” US9851368B2. WJG, EY, YW and ST are shareholders in CholesteniX Ltd., (© 2023 The Authors.)

Most biologically active oxysterols have a 3β-hydroxy-5-ene function in the ring system with an additional site of oxidation at C-7 or on the side-chain. In blood plasma oxysterols with a 7α-hydroxy group are also observed with the alternative 3-oxo-4-ene function in the ring system formed by ubiquitously expressed 3β-hydroxy-Δ 5 -C 27 -steroid oxidoreductase Δ 5 -isomerase, HSD3B7. However, oxysterols without a 7α-hydroxy group are not substrates for HSD3B7 and are not usually observed with the 3-oxo-4-ene function. Here we report the unexpected identification of oxysterols in plasma derived from umbilical cord blood and blood from pregnant women taken before delivery at 37+ weeks of gestation, of side-chain oxysterols with a 3-oxo-4-ene function but no 7α-hydroxy group. These 3-oxo-4-ene oxysterols were also identified in placenta, leading to the hypothesis that they may be formed by a previously unrecognized 3β-hydroxy-Δ 5 -C 27 -steroid oxidoreductase Δ 5 -isomerase activity of HSD3B1, an enzyme which is highly expressed in placenta. Proof-of-principle experiments confirmed that HSD3B1 has this activity. We speculate that HSD3B1 in placenta is the source of the unexpected 3-oxo-4-ene oxysterols in cord and pregnant women's plasma and may have a role in controlling the abundance of biologically active oxysterols delivered to the fetus.

The aim of this study was to identify oxysterols and any down-stream metabolites in placenta, umbilical cord blood plasma, maternal plasma and amniotic fluid to enhance our knowledge of the involvement of these molecules in pregnancy. We confirm the identification of 20S-hydroxycholesterol in human placenta, previously reported in a single publication, and propose a pathway from 22R-hydroxycholesterol to a C 27 bile acid of probable structure 3β,20R,22R-trihydroxycholest-5-en-(25R)26-oic acid. The pathway is evident not only in placenta, but pathway intermediates are also found in umbilical cord plasma, maternal plasma and amniotic fluid but not non-pregnant women., Competing Interests: WG and YW are listed as inventors on the patent “Kit and method for quantitative detection of steroids” US9851368B2. WG, EY, and YW are shareholders in CholesteniX Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dickson, Yutuc, Thornton, Wang and Griffiths.)

Alterations in brain cholesterol homeostasis have been broadly implicated in neurological disorders. Notwithstanding the complexity by which cholesterol biology is governed in the mammalian brain, excess neuronal cholesterol is primarily eliminated by metabolic clearance via cytochrome P450 46A1 (CYP46A1). No methods are currently available for visualizing cholesterol metabolism in the living human brain; therefore, a noninvasive technology that quantitatively measures the extent of brain cholesterol metabolism via CYP46A1 could broadly affect disease diagnosis and treatment options using targeted therapies. Here, we describe the development and testing of a CYP46A1-targeted positron emission tomography (PET) tracer, 18 F-CHL-2205 ( 18 F-Cholestify). Our data show that PET imaging readouts correlate with CYP46A1 protein expression and with the extent to which cholesterol is metabolized in the brain, as assessed by cross-species postmortem analyses of specimens from rodents, nonhuman primates, and humans. Proof of concept of in vivo efficacy is provided in the well-established 3xTg-AD murine model of Alzheimer's disease (AD), where we show that the probe is sensitive to differences in brain cholesterol metabolism between 3xTg-AD mice and control animals. Furthermore, our clinical observations point toward a considerably higher baseline brain cholesterol clearance via CYP46A1 in women, as compared to age-matched men. These findings illustrate the vast potential of assessing brain cholesterol metabolism using PET and establish PET as a sensitive tool for noninvasive assessment of brain cholesterol homeostasis in the clinic.