Studies of Rat Brain Metabolism Using Proton Nuclear Magnetic Resonance: Spectral Assignments and Monitoring of Prolidase, Acetylcholinesterase, and Glutaminase

Abstract

The first application of inversion-recovery spin-echo proton nuclear magnetic resonance spectroscopy to the monitoring of reactions in rat brain preparations is presented. The initial report of the assignment of proton spin-echo nuclear magnetic resonance spectra from rabbit brain homogenates (C. R. Middlehurst et al., J. Neurochem. 42, 878-879, 1984) was used to assist in the assignment of spectra acquired from rat brain homogenates that were obtained from animals killed by cervical fracture or focussed microwave irradiation. Microwave-irradiated brains were divided into four major anatomical regions. Differences in metabolite levels were detected when spectra from fresh tissue and from various regions were compared. The in situ steady-state kinetics of prolidase in whole brain homogenate was determined. The procedure relies on the spectral differences between enzyme substrates and reaction products. The concentration dependence of the rate of hydrolysis of glycyl-L-proline was discribable by the Michaelis-Menten expression with a Michaelis constant of 1.90 mmol L-1 and a maximal velocity of 9.30 mumol min-1 mg-1 protein. The reactions catalysed by glutaminase and acetylcholinesterase in the brain were also monitored.

Proton NMR chemical shifts and coupling constants for brain metabolites

“…Its presence has been reported in several cell culture and extract studies, though its abundance was questioned as a possible artifact of the sample preparation. 11 Nevertheless, it has been observed in high-resolution NMR studies of normal rat brain, 30 human resected tissues, 31 and cell cultures, 32 and to be present at a high concentration in neonatal rat cerebellum with a progressive decrease with age. 33 Concentrations in human brain are approximately 0.4-0.8 mmol/kg ww , 31 with increased levels observed in brain tumors, 34 ischemia, 35 and in vivo in brain abscesses.…”

Cerebral White Matter in the Centrum Semiovale Exhibits a Larger N‐acetyl Signal than Does Gray Matter in Long Echo Time 1H‐Magnetic Resonance Spectroscopic Imaging

“…Later work suggested that other brain constituents having N-acetyl moieties (e.g., N-acetylaspartylglutamate), also contribute to signal intensity in this region of the spectrum (5,6). Published evidence also shows that glutamate, glutamine, and various macromolecular constituents contribute to this spectral region (4,(7)(8)(9)(10)(11)(12), although it is unlikely that these latter signals are present in long echo time spectra, of the sort we present here, due to T, and J-modulation losses (12). Despite the controversy, most investigators accept the notion that NAA is a major contributor to the 2.02 ppm signal.…”

“…'H-MRSI is not without disadvantages. High resolution 2-dimensional phase encoding is time consuming and performing studies with TR values that are [5][6][7][8][9][10] times the longest in vivo T, values are usually not practical. Consequently, 'H-MRSI data are often moderately TI-weighted.…”

Assimilation of α-glutamyl-peptides by human erythrocytes. A possible means of glutamate supply for glutathione synthesis

“…Glutaminase activity can be readily monitored by using 400MHz 1H-n.m.r. since the glutamine and glutamate Hy protons have significantly different chemical shifts (Middlehurst et al, 1984). Incubation of rabbit or human erythrocyte lysates or whole cell suspensions (haematocrit 0.70-0.80) with glutamine (5 mmol/l) led to no detectable production of glutamate (i.e.…”

“…Proton spin-echo n.m.r. spectroscopy was used in these studies because of its now well-documented ability to monitor the kinetics of peptide transport and hydrolysis in situ (King et al, 1983;Middlehurst et al, 1984).…”