Metabolomics of Rat Brain After Treatment with Phenelzine: High-Resolution Mass Spectrometric Demonstration of Increased Brain Levels of N-Acetyl Amino Acids

Background: Phenelzine (PLZ) is a non-specific monoamine oxidase inhibitor that has demonstrated
clinical efficacy in patients with treatment resistant depression. The mechanism of action with regard to this
efficacy is complicated in that its metabolite, β-phenylethylidenehydrazine (PEH), is an inhibitor of amino
acid transaminases resulting in dramatic brain elevations of GABA, alanine, ornithine and tyrosine. The full
neurochemical profile of PLZ and PEH remain to be explored.
Objective: To undertake a non-targeted metabolomics study of phenelzine on rat brain neurochemistry.
Methods: We undertook a high-resolution mass spectrometric metabolomics analysis of rat cortical brain
1 and 12 hours after intraperitoneal dosing with PLZ or PEH. Tandem mass spectrometry was utilized to
obtain relative quantitation data.
Results: N-acetyl amino acids were found to be elevated in cortical brain tissue following either PLZ or
PEH treatments.
Conclusions: Our data indicate PLZ treatment significantly augments brain levels of N-acetyl amino acids
and that this may involve inhibition of deacylases by PEH and/or induction of N-amino acid
acetyltransferases.



Introduction
Phenelzine is a nonspecific monoamine oxidase (MAO) inhibitor that has been utilized in patients with treatment resistant depression [1][2][3][4]. Phenelzine is a potent inhibitor of both MAO-A and MAO-B which increases brain monoamine levels. In contrast, its metabolite, βphenylethylidenehydrazine (PEH), is a weak MAO inhibitor but is a potent transaminase inhibitor that significantly elevates brain levels of GABA and alanine, tyrosine, and ornithine [5][6][7][8]. PLZ and PEH also are both carbonyl scavenging agents that reduce reactive aldehyde toxicity in vivo and in vitro. The aldehyde scavenging actions of PLZ and PEH have been hypothesized to be responsible for their neuroprotective actions in animal models of ischemia-reperfusion injury, traumatic brain injury, spinal cord injury, experimental autoimmune encephalitis, and in vitro aldehyde toxicity [9][10][11][12][13][14]. These data indicate that PLZ and PEH have very complex pharmacodynamic effects. To further investigate the biochemical effects of these compounds, we undertook a non-targeted metabolomics study of PLZ and PEH on the rat brain metabolome. This study revealed that PLZ and PEH significantly augment brain levels of free N-acetyl amino acids.

I Rat Brain Samples
Male Sprague Dawley rats were dosed intraperitonially with 30 mg/kg of PLZ or PH, and the brains harvested after decapitation at 1 and 12 hours. The brains were frozen immediately in isopentane on solid carbon dioxide and then removed to containers stored at -80 o C until the frontal cortex was dissected out for metabolomics analysis. All procedures Neurology  involving animals were approved by the University of Alberta Biosciences Animal Care and Use Committee (AUP00000216) and were in accordance with the guidelines of the Canadian Council on Animal Care.

III High-Resolution Mass Spectrometric Analyses
Samples underwent flow infusion analyses at a flow rate of 12 μL per min. and were analysed via high-resolution mass spectrometry (HR-MS) utilizing a Q-Exactive benchtop orbitrap (Thermo Fisher) with a resolution of 140,000 and less than 1 ppm mass error. Negative ion electrospray ionization (NESI) with a sheath gas of 12, a spray voltage of 3.7 kV, and a capillary temperature of 321 o C was used. For the pilot metabolomics analysis, the scan was from 60 to 900 amu. The data were analysed via an in-house Excel (Microsoft) spreadsheet with over 1200 metabolites of interest.
To obtain relative quantitative data of N-acetyl amino acids, MS 2 studies utilized a window of 0.4 amu for the precursor ion and the product ions were acquired at high resolution (< 1 ppm mass error). For MS 2 studies ( Table 1) the neutral collision energy (NCE) was optimized between 20 and 30 eV.

IV Data Presentation
Data are presented as relative (R) N-acetyl amino acid levels (i.e. the ratio of the signal intensity of the endogenous N-acetyl amino acid to the signal intensity of an appropriate stable isotope internal standard), corrected for protein, ± SD (N=5).

Results
The most marked observation from our preliminary non-targeted metabolomics analysis was increased brain levels of N-acetyl amino acids with PLZ and PEH dosing. Since this was a flow infusion analysis, there were a number of potential isobars with the exact mass of each Nacetyl amino acid. Therefore, to obtain relative quantitation data we performed MS 2 analyses. This approach clearly demonstrated increased levels of an array of brain N-acetyl amino acids ( Figure 1). It is interesting to note that N-acetylaspartate levels, which are in millimolar (mM) concentrations in the brain, were unaffected by the drug treatments.
The functional roles of free N-acetyl amino acids remain to be more fully elucidated but decreased plasma levels of N-acetylmethionine have been monitored in cystic fibrosis patients and childhood obesity, and decreased plasma levels of N-acetylglycine in obesity [30][31][32][33]. Cadmium, which is an ACY1 inhibitor, elevates urinary levels of Nacetylglutamate, N-acetylglutamine, and N-acetylphenylalanine, suggesting a rapid turnover rate for these N-acetyl amino acids in vivo [34,35]. Similarly, precursor labeling studies have defined the rapid dynamics of N-acetylmethionine synthesis in human oligodendrocyte cultures [22].
A detailed analysis of N-acetyl amino acids in dogs with gallbladder mucocele formation found decreased blood levels of N-acetylated alanine, glycine, glutamate, isoleucine, leucine, methionine, serine, and threonine [35]. In contrast, the bile in these dogs was characterized by increased levels of N-acetylated glutamate, histidine, isoleucine, leucine, lysine, threonine, tryptophan, tyrosine, and valine. These data suggest that N-acetyl amino acids play a complex metabolic function in the gallbladder.
With regard to brain function, free N-acetylmethionine, Nacetylglutamine, N-acetylglutamate, N-acetylasparagine, and Nacetylalanine have all been monitored in the human brain [23,[36][37][38][39][40]. N-acetylglutamate is a critical modulator of the urea cycle, while Nacetylleucine modulates the neuronal activity of vestibulocerebellar and posterolateral thalamic circuits involved in vestibular function, while Nacetylglutamine appears to be involved in the sleep-wake cycle [40][41][42]. Clearly, we currently have limited knowledge of the roles of N-acetyl amino acids in brain function.

Conflicts of Interest
None.