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 protonmagnetic resonance spectroscopy to occur exclusively at die 4 position of the indole nucleus. The antibody reacted with a variety of N-acetylated indoles, and absolute specificity was dependent upon 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 [ 2 H 3 ]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 weeks 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. (Endocrinology 101: 119, 1977)

Plasma melatonin profiles of Romney Marsh sheep in natural photoperiod and in acutely extended darkness

“…Just how melatonin performs this function is unclear. In sheep and some other species the secretion of melatonin has generally been reported to follow the duration of the dark period (Rollag & Niswender, 1976;Kennaway et al, 1977;Lincoln & Short, 1980;Arendt et al, 1981) and this mechanism is believed to underlie the seasonal repro¬ ductive response given the longer period of melatonin secretion in winter than summer (Maywood et al, 1990). However, another major hypothesis is centred around the possibility that there is a phase of the circadian cycle sensitive to the presence of melatonin (Tamarkin et al, 1976;Rollag et al, 1978) …”

Plasma melatonin in ewes after ovariectomy

“…Evidence of pineal involvement in the ovine reproductive cycle has also been furnished by measurement of peripheral melatonin levels. Circulating melatonin in the ewe shows a diurnal rhythm, values being elevated during darkness and low in continuous light (Rollag & Niswender, 1976; Kennaway, Frith, Phillipou, Matthews & Seamark, 1977). Moreover, the duration of melatonin secretion may reflect the length of darkness (Rollag, O'Callaghan & Niswender, 1978).…”

Circulating Levels of Melatonin following Its Oral Administration or Subcutaneous Injection in Sheep and Goats

“…More recently melatonin has been implicated in the control of seasonal gonadal regression in hamsters (Tamarkin et al 1976). These findings, together with the development of radioimmunoassays for circulating blood levels of melatonin in many species (Arendt et al 1975; Rollag andNiswender 1976;Kennaway et al 1977), have resulted in an upsurge in interest in how melatonin and the pineal gland exert their influence on gonadal function. Many of the early experiments in which injection of melatonin was used to evoke physiological or endocrine changes can be criticized not only on the basis of administration at inappropriate times (Tamarkin et al 1976) but also on the basis that pharmacological rather than physiological doses of melatonin were used (Minneman and Wurtman 1975).…”

“…Plasma melatonin was determined by a radioimmunoassay (Kennaway et al 1977). After the addition of borate buffer (0·5 M; pH 10; 1 ml), 0 ·1-2 ml of plasma was extracted with dichloromethane and the organic extract chromatographed on Lipidex 5000 columns with dichloromethane:hexane (1: 1 v/v) as eluant.…”