Sensitive assays for the determination of monoamine oxidase and phenol sulphotransferase activity in small tissue samples

Abstract

A method to assay phenol Sulphotransferase (PST) and monoamine oxidase (MAO) in brain (anterior pituitary gland, hypothalamus) and liver specimens as small as 4 mg is described. The specimens were homogenized (sonicated) in various volumes of a buffer, the smallest being 100 μL, to obtain the homogenates. MAO assay was carried out using 30 μL of the homogenate and for PST assay, 30 μL of either the homogenate or, in the case of liver, the supernatant (100,000 × g for 60 min). The radiolabeled products of the enzymatic reactions were separated from the radiolabeled substrates by high-pressure liquid chromatography (HPLC) and the radioactivity of the eluted products measured directly by a radioisotope detector coupled to the HPLC system. The constraint of the assay protocol was not the weight of the specimens but the volume of buffer used in the preparation of the homogenate. Although 100 μL was a convenient working volume, the tissue can also, with care, be sonicated in a 50 μL buffer. With extremely small specimens, weighed fractions of the specimens could be sonicated directly in the control and experimental incubation mixtures bypassing the preparation of the homogenate. Thus, the overall method offers, for the first time, a reliable and adaptable means for measuring MAO and PST in small to extremely small tissue specimens.