High performance liquid chromatography-electrospray ionization mass spectrometric (LC-ESI-MS) methodology for analysis of amino acid energy substrates in microwave-fixed microdissected brain tissue

•Hypoglycemic reductions in brain glutamine (Gln) and glutamate (Glu) imply use as alterative metabolic fuels.•Ventromedial hypothalamic nucleus (VMN) Gln, Glu, and aspartate (Asp) content was measured after microwave fixation.•Amino acid reaction with FMOC-Cl is time-dependent.•Chromatographic reso...

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Published in:	Journal of pharmaceutical and biomedical analysis 2020-05, Vol.184, p.113123-113123, Article 113123
Main Authors:	Bheemanapally, Khaggeswar, Ibrahim, Mostafa M.H., Briski, Karen P.
Format:	Article
Language:	eng
Subjects:	9-fluorenylmethyl Amino Acids - metabolism Animals Aspartate Brain - metabolism Chloroformate Chromatography, High Pressure Liquid - methods Glucose - metabolism Glutamine Insulin-induced hypoglycemia Male Microdissection - methods Microwaves Rats Rats, Sprague-Dawley Spectrometry, Mass, Electrospray Ionization - methods Ventromedial hypothalamic nucleus
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Summary:	•Hypoglycemic reductions in brain glutamine (Gln) and glutamate (Glu) imply use as alterative metabolic fuels.•Ventromedial hypothalamic nucleus (VMN) Gln, Glu, and aspartate (Asp) content was measured after microwave fixation.•Amino acid reaction with FMOC-Cl is time-dependent.•Chromatographic resolution of FMOC amino acid derivatives occurs less than 7 min.•Gln and Asp were decreased by acute hypoglycemia, but affected differently by chronic exposure. Hypoglycemia deprives the brain of its primary energy source glucose. Reductions in whole-brain amino acid energy substrate levels suggest that these non-glucose fuels may be metabolized during glucose shortage. Recurring hypoglycemia can cause mal-adaptive impairment of glucose counter-regulation; yet, it is unclear if amplified reliance upon alternative metabolic substrates impedes detection of continuing neuro-glucopenia. This research aimed to develop high-sensitivity UHPLC-electrospray ionization mass spectrometric (LC-ESI-MS) methodology, for complementary use with high-neuroanatomical resolution microdissection tools, for measurement of glucogenic amino acid, e.g. glutamine (Gln), glutamate (Glu), and aspartate (Asp) content in the characterized glucose-sensing ventromedial hypothalamic nucleus (VMN) during acute versus chronic hypoglycemia. Results show that VMN tissue Gln, Glu, and Asp levels were significantly decreased during a single hypoglycemic episode, and that Gln and Asp measures were correspondingly normalized or further diminished during renewed hypoglycemia. Results provide proof-of-principle that LC-ESI-MS has requisite sensitivity for amino acid energy substrate quantification in distinctive brain gluco-regulatory structures under conditions of eu- versus hypoglycemia. This novel combinatory methodology will support ongoing efforts to determine how amino acid energy yield may impact VMN metabolic sensory function during persistent hypoglycemia.
ISSN:	0731-7085 1873-264X