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.
This study investigates the ability of a 1 h light pulse of different intensities at night to suppress plasma melatonin in goats. Six female Saanen dairy goats, about 2 yr old, were housed in a light-tight shed. The goats were habituated for 1 wk to an 8L:16D photoperiod (40.70 +/- 4.16 microW/cm2; 137 +/- 14 lux), lights on 0800 h. A 1 h light pulse, of different intensity on each occasion, was given from 1900 to 2000 h. Light intensity was measured by using a lux meter (mean of 36 measurements at goat's eye level). Five different light intensities were given during December in the order 4.22 +/- 0.62 microW/cm2 (14.2 +/- 2.1 lux), 0.68 +/- 0.09 microW/cm2 (2.3 +/- 0.3 lux), 0.26 +/- 0.004 microW/cm2 (0.87 +/- 0.14 lux), darkness, 40.70 +/- 4.16 microW/cm2 (137 +/- 14 lux), with 1-3 d between treatments. The goats were bled hourly from 1500 to 1900 h and every 15 min from 1900 to 2100 h, and a last bleed occurred at 2200 h. Dark-phase samples were taken in dim red light (less than 0.03 microW/cm2; 0.1 lux). Plasma was assayed for melatonin by radioimmunoassay. Suppression of melatonin concentrations increased as light intensity increased as follows: Darkness, 0%; 0.26 +/- 0.004 microW/cm2; 0%; 0.68 +/- 0.09 microW/cm2; 43.1%; 4.22 +/- 0.62 microW/cm2, 71.1%; 40.70 +/- 4.16 microW/cm2, 81.2%. Suppression was significant (P less than 0.05) at light intensities greater than 0.68 microW/cm2, 2.3 lux. A hyperbolic relationship existed between percent suppression and light intensities.
Summary
Melatonin signals night time by its rhythmic profile of secretion. Its measurement provides the best indicator of internal circadian clock timing. Treatment with melatonin induces sleepiness and lowers body temperature during ‘biological day’ (i.e. when its endogenous secretion is minimal) and it acts as a ‘chronobiotic’ to change the timing of the circadian clock. It has been used successfully to treat disorders of biological rhythms in humans
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