Adrenal Hormone Interactions and Metabolism: A Single Sample Multi-Omics Approach

Abstract

The adrenal gland is important for many physiological and pathophysiological processes, but studies are often restricted by limited availability of sample material. Improved methods for sample preparation are needed to facilitate analyses of multiple classes of adrenal metabolites and macromolecules in a single sample. A procedure was developed for preparation of chromaffin cells, mouse adrenals, and human chromaffin tumors that allows for multi-omics analyses of different metabolites and preservation of native proteins. To evaluate the new procedure, aliquots of samples were also prepared using conventional procedures. Metabolites were analyzed by liquid-chromatography with mass spectrometry or electrochemical detection. Metabolite contents of chromaffin cells and tissues analyzed with the new procedure were similar or even higher than with conventional methods. Catecholamine contents were comparable between both procedures. The TCA cycle metabolites, cis-aconitate, isocitate, and α-ketoglutarate were detected at higher concentrations in cells, while in tumor tissue only isocitrate and potentially fumarate were measured at higher contents. In contrast, in a broad untargeted metabolomics approach, a methanol-based preparation procedure of adrenals led to a 1.3-fold higher number of detected metabolites. The established procedure also allows for simultaneous investigation of adrenal hormones and related enzyme activities as well as proteins within a single sample. This novel multi-omics approach not only minimizes the amount of sample required and overcomes problems associated with tissue heterogeneity, but also provides a more complete picture of adrenal function and intra-adrenal interactions than previously possible.

Metabolomics in paraganglioma: applications and perspectives from genetics to therapy

“…Recently, we reported a methodological improvement of LC-MS/MS mediated tissue metabolomics, by showing that a commercial buffer enables analysis by multiple targeted and untargeted LC-MS/MS assays, as well as RNA and protein analysis, including enzyme activities, from the same piece of tissue (Bechmann et al, 2021). Other groups have also published on simultaneous tissue extraction methods for multi-omics focusing on proteins or RNA in combination with metabolites and lipids (Coman et al, 2016, Leuthold et al, 2018, Kang et al, 2021.…”

“…In the future, metabolomic assessment of patients with PPGL, in tumor tissue and blood, will most likely move towards broader metabolic signatures using bigger targeted panels or untargeted approaches that will be combined with genomic and possibly transcriptomic or proteomic information. In this respect, the methods for efficient extraction of different molecules from the same piece of tumor become very important (Bechmann et al, 2021). NMR-based untargeted metabolomics studies have so far only been performed on a very limited number of PPGL tissues.…”

Isolation and in vitro cultivation of adrenal cells from mice

“…(2018) . Catecholamine production in the cells can be measured using mass spectrometry as described previously ( Bechmann et al., 2021b ). In our experience, the medullary cells divide and grow faster than the cortical cells ( Figure 7 ).…”

HIF2α regulates the synthesis and release of epinephrine in the adrenal medulla

“…Adrenal glands were incubated in disruption buffer (component of Invitrogen™ Paris™ Kit, AM 1921, Thermo Fisher Scientific) for 15 min at 4 °C, homogenized in a tissue grinder, followed by incubation for 15 min on ice and further preparation [ 31 ]. Adrenal catecholamines , norepinephrine, epinephrine, and dopamine were measured by high pressure liquid chromatography (HPLC) coupled with electrochemical detection after batch extraction with alumina, as previously described [ 32 ].…”