Isolation of Organic Anions by Extraction with Liquid Anion Exchangers and Its Application to Micromethods for Acetylcholinesterase and 4-Aminobutyrate Aminotransferase

Abstract

Organic anions of particular importance to biochemistry such as Krebs cycle intermediates, glycolysis intermediates, simple fatty acids, adenine nucleotides and CoA derivatives can be quantitatively extracted from a buffered solution by high-molecular-weight ammonium salts in an organic solvent. Phosphate salts of tertiary amines in chloroform were the most efficient extractants. The isolation procedure was found to be an example of amine neutralization. The effect of pH, different inorganic anions, volume ratios between the two phases, concentration of the isolated anions and concentration of the ammonium salts have been investigated. The extraction technique has been applied to rapid and sensitive radiochemical methods for the determination of acetylcholinesterase and 4-aminobutyrate aminotransferase activities.Previously, we have demonstrated that sodium tetraphenylboron (Kalignost) in heptanone or alkylnitriles constitutes a powerful liquid cation exchanger [I]. The complex has successfully been used in the isolation of several cations such as acetylcholine or aromatic amines from aqueous solutions [l] and has made it possible to develop highly sensitive radiochemical enzyme assays for measurements of choline acetyltransferase

Monocarboxylate transport in erythrocytes

“…Difficulties may also result from decomposition of the labeled anion [91]. Adequate chromatographic [49] or other analytical techniques [95] have to be used to cope with these problems.…”

Acute stress and GABAergic function in the rat brain

“…The method described by Tunniciff & Smith (1981) was employed with slight modifications (Otero Losada, 1988 (Sterri & Fonnum, 1978), and the radioactivity in the organic phase was determined by scintillation counting.…”

β‐phenylethylidenehydrazine, a novel inhibitor of GABA transaminase

“…Analysis was performed using a WISP 710B automatic injection system (Waters, Milford, MA) and a 5µ ODS 2 column (Phenomenex, Torrance, CA) linked to a Waters Fluorescence detector (Model 420-AC) with an HP 3392A integrator. GABA-transaminase (GABA-T) activity was measured using a procedure modified from Sterri and Fonnum [1978]. MAO-A and -B activities were measured using a radiochemical assay protocol adapted from Lyles and Callingham [1982]; 14 C-labeled 5-hydroxytryptamine (5-HT) and β-phenylethylamine were used as substrates for MAO-A and -B, respectively.…”