A Specific Radioimmunoassay for Melatonin in Biological Tissue and Fluids and its Validation by Gas Chromatography-Mass Spectrometry

Abstract

A specific practical radioimmunoassay suited to determinations of melatonin in both tissues and body fluids is described. The rabbit antibody employed was raised to an antigen formed by condensation between N-acetylserotonin and the Mannich adduct of bovine serum albumin and formaldehyde. Substitution was shown by PMR spectroscopy to occur exclusively at the 4 position of the indole nucleus. The antibody reacted with a variety of N-acetylated indoles, and absolute specificity was dependent on the extraction procedure and column (Lipidex 5000) chromatography. In addition to the usual reliability criteria, the validity of the assay was checked by gas chromatography-mass spectrometry using [2H3]melatonin as an internal standard, the preparation of which is described. The occurrence of melatonin in the plasma of man, sheep, rat and chicken was confirmed, and its presence in the plasma of the pig (22-76 pg/ml), donkey (24-128 pg/ml), cow (20-320 pg/ml), camel (29-221 pg/ml) and a scincid lizard (20-500 pg/ml) established. A nocturnal rise in plasma melatonin content occurred in all species. Melatonin was found in the plasma of ewes 2-12 wk after pinealectomy, but the nocturnal rise was abolished. The results establish a nyctohemeral variation in plasma melatonin in a wide variety of species, and indicate that sources of melatonin other than the pineal may assume precedence following pinalectomy.