DEVELOPMENT OF A SPECIFIC EXTRACTED RADIOIMMUNOASSAY FOR METHIONINE ENKEPHALIN IN HUMAN PLASMA AND CEREBROSPINAL FLUID

Abstract

A sensitive extracted radioimmunoassay for methionine enkephalin (met-enkephalin) has been developed which allows measurement of its concentration in human plasma and cerebrospinal fluid (CSF). The first evidence for the existence of met-enkephalin in the circulation of man is described and its presence in CSF confirmed. The susceptibility of the methionine residue in met-enkephalin to undergo oxidation to the methionine sulphoxide analogue was utilized. All extracted samples were oxidized by hydrogen peroxide before assay and this allowed measurement of total met-enkephalin. The assay had unique specificity with no cross-reaction with leucine enkephalin, purified human β-endorphin or β-lipotrophin (β-LPH). A purification method for radio-iodinated enkephalin has been developed with octadecasilyl-silica (ODS-silica) (10 μm) yielding a high-affinity monoiodinated tracer stable on storage for up to 3 months at 4 °C. A method to extract met-enkephalin from acidified plasma or CSF has been developed with larger particle size ODS-silica (35–70 μm) suitable for extracting repeated samples. Met-enkephalin immunoreactivity was detectable in plasma of all subjects tested and ranged from 14 to 140 pg/ml. In CSF, however, the range was 5–29 pg/ml. Met-enkephalin immunoreactivity was not generated by incubating exogenous or endogenous β-LPH and β-endorphin in plasma or CSF. Two hypopituitary subjects and one dexamethasone-suppressed subject, all with undetectable immunoreactive plasma adrenocorticotrophic hormone and NH₂- and CO₂H-terminal β-LPH, had measurable met-enkephalin in their plasma suggesting met-enkephalin was not of pituitary origin nor a breakdown product of secreted β-LPH or β-endorphin.