D J Skene, I Smith, J Arendt

A sensitive, specific, reproducible and practical radioimmunoassay for the determination of 5-methoxytryptophol (ML) in pineal glands of different species has been developed. High-affinity specific antisera were produced by immunization of sheep with ML-bovine serum albumin. Iodinated ML, used as the radiolabel, was synthesized by direct iodination of ML using 1, 3, 4, 6, -tetrachloro-3, 6-diphenylglycouril as the oxidant. Sensitivity of the assay was 0.005 pmol/tube. The validity of the assay was checked using classical techniques. Cross-reactivity with other indoles was negligible. Parallel inhibition curves were obtained for rat, hamster, sheep and tortoise pineal homogenates. Using thin-layer chromatography, tortoise pineal immunoreactivity also co-chromatographed with standard ML. Samples (n = 5) with ML concentrations of 0.013, 0.052 and 0.209 pmol/tube had intra-assay coefficients of variation of 9.8, 5.7 and 7.6% respectively. Their respective interassay coefficients of variation were 17.7, 16.5 and 11.4% (n = 8). The pineal concentration of ML was found to be species dependent. Afternoon ML levels were 0.052 +/- 0.002 (S.E.M.) pmol/gland in the rat (n = 16), 0.539 +/- 0.089 pmol/gland in the hamster (n = 16), 1.73 +/- 0.225 nmol/g in the sheep (n = 10) and 7.15 +/- 0.465 pmol/gland in the tortoise (n = 4). The ratio of ML:melatonin content in the pineal gland also showed a large interspecies variation with values of 0.02 in the rat, 0.22 in the sheep, 2.7 in the hamster and 17 in the tortoise.