The effect of anticoagulants, enzyme inhibitors and long term storage on the extraction of known concentrations of methionine enkephalin in human plasma

Abstract

The stability of methionine enkephalin (M-E) during long term storage was investigated using various anticoagulants and enzyme inhibitors, eg EDTA, heparin, trasylol, citric acid. Plasma was stored for different lengths of time up to six weeks. High pressure liquid chromatography (HPLC) was used to separate and quantify M-E. We found that EDTA, heparin or trasylol per se are ineffective in preserving M-E for short term extraction. Blood collected in chilled heparin tubes with citric acid crystals and the plasma further acidified with hydrochloric acid gave the highest recovery. With storage times up to six weeks further degradation was marked in samples taken in plain tubes but did not occur with tubes containing citric acid crystals and hydrochloric acid. Enkephalins normally occur in biological tissues and fluids in very low concentration pmo1/g or m1 1,2. The source of M-E in blood is so far unknown. There are speculations that it could be from the adrenal medulla, 3,4 and the ovary 5. Enkephalins are known to influence a variety of physiological processes. Among these are their effects on pain, respiration, blood pressure, behaviour, temperature, food intake 6,7 and the secretiion of several pituitary hormones 8,9,10. They may act as neurotransmitters of neuromodulators 11. It is well known that the physiological activity of enkephalins is transitory with a half life in vitro of two minutes due to their rapid degradation by proteolytic enzymes. Consequently the quantitative analysis of enkephalins is inaccurate unless effective methods to inhibit these enzymes are employed. To minimise the effect of inter-assay variation, analysis is preferably of large batches and long-term storage of specimens is required. The purpose of this study is to compare the effect of long term storage following the use of various agents to inhibit enzyme activity.