Radioreceptor-assayによるヒト髄液中methionine-enkephalin様物質の測定

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In recent years it has been stated that endogenous morphine-like substances, opioid peptides, exist in the brain and the pituitary gland. Two pentapeptides with a morphine-like activity, methionine (met) - and leucine-enkephalin, are distributed in the whole brain and highly concentrated in the basal ganglia. Another class of the opioid peptides of a larger molecular weight, α-, β- and γ-endorphin, is mainly found in the pituitary gland. Based on the different distributions, it has been generally supposed that enkephalin (s) may act as neurotransmitters and that endorphins may act as hormones. The measurement of these peptides in human cerebrospinal fluid (CSF) and plasma would be of great use in ascertaining the physiological significance of these peptides as well as their possible involvement in such clinical symptoms as intractable pain. The purpose of this study was to establish a radioreceptor-assay system of the met-enkephalin-like substance in human CSF. A particulate fraction was prepared from rat brain essentially according to the method of Pasternak and used as a receptor. In order to obtain the most sensitive radioreceptor-assay, displacement curves by met-enkephalin were compared with each other using three kinds of radiolabeled ligand : 3H-met-enkephalin, 3H-naloxone and 3H-dihydromorphine. When 3H-dihydromorphine was used as the radiolabeled ligand, the concentration of met-enkephalin required to inhibit 50% of specific binding (IC50) was the lowest. The addition of 1mM EDTA and 2mM Mg was found to decrease further the IC50 and enhance the binding. Thus the radioreceptor-assay of the met-enkephalin-like substance in CSF was carried out using 3H-dihydromorphine as the radiolabeled ligand in the presence of 1 rnM EDTA and 2mM Mg. The sensitivity of the assay ranged from about 1 to 100 pmoles of met-enkephalin. The isolation of met-enkephalin from CSF was performed by a Sephadex G 10 gel filtration followed by a SP-Sephadex (H+) column chromatography. Large molecular substances such as β-endorphin were separated from the met-enkephalin by gel filtration. Sodium and non-specific inhibitor (s) of the specific binding of 3H-dihydromorphine were removed from CSF by the chromatography. Five ml of CSF were applied to two successive columns, and the elutes were lyophilized and subjected to quantification in the radioreceptor-assay. The overall recovery of met-enkephalin was about 60%. Human CSF was obtained from 8 patients hospitalized for neurosurgical study and therapy. Ten ml of CSF were drawn by lumbar puncture and kept frozen at -60°C until assayed. All assays were duplicated. The met-enkephalin-like substance levels were 3.3 ± 2.1 (mean ± S.D., n=8) pmoles/ml and ranged from 0.7 to 6.7 pmoles of the met-enkephalin. equivalents.
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Radioreceptor-assayによるヒト髄液中methionine-enkephalin様物質の測定

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