Abstract. A new radioimmunoassay (RIA) using [125I]melatonin as tracer for determination of melatonin in biological fluids has been developed. Melatonin antisera were raised in rabbits by immunization with bovine thyroglobulin conjugate of n-acetyl-5-methoxytryptophan. Two high-affinity and specific antisera were obtained. Unlike in previous studies melatonin was radioiodinated directly. Iodo-Gen was used as the oxidant. Radioiodinated melatonin was purified by TLC for use in RIA. Melatonin was extracted from plasma, serum and urine samples (1 ml) with chloroform. Using the extraction the sensitivity of the RIA method was 18 fmol/ml of original sample. Plasma, serum and urine extracts diluted parallelly with synthetic melatonin in RIA. HPLC analysis of plasma and serum extracts showed only one immunoreactive peak co-eluting with synthetic melatonin. Majority of urine immunoreactivity co-eluted with synthetic melatonin, but 7–23% contaminating immunoreactivity was also observed. Daytime values for rat plasma, human serum and urine melatonin were 30–60, 20–40 and 50–130 fmol/ml and the respective night values were 160–300, 180–370 and 230–470 fmol/ml. Thus a characteristic diurnal rhythm of melatonin was observed in all cases. The urinary excretion of immunoreactive melatonin during the day was 3–9 and during the night 11–28 pmol/h. Thus we have developed a specific and valid RIA method for the determination of plasma and serum melatonin. Despite the incomplete specificity of the RIA for urine determinations, a clear diurnal rhythm for urine melatonin was observed. The distinct advantage of the utilization of [125I]melatonin as tracer is that the costly and cumbersome scintillation counting can be avoided.
Olli Vakkuri, Juhani Leppäluoto and Olli Vuolteenaho
Olli Vuolteenaho, Juhani Leppäluoto, Matti Höyhtyä and Jorma Hirvonen
The presence of β-endorphin in human pituitary was studied using radioimmunoassay, opiate receptor binding assay and different chromatographic methods. Adult human pituitaries were found to contain 291 ± 59 μg/g β-endorphin immunoreactivity, of which 15–45% eluted like β-endorphin in Sephadex G-75 gel filtration. The yield of β-endorphin immunoreactivity was found to be dependent of the extraction method used, but the relative proportion of the different immunoreactive components (proopiomelanocortin, β-lipotrophin and β-endorphin) was approximately identical irrespective of the extraction method. No correlation was observed between the time lag from death to autopsy (within 7 days) and either the total amount of β-endorphin immunoreactivity or the β-lipotrophin/β-endorphin ratio in the acid extracts of human pituitaries. Repeated freezing and thawing was found to cause a progressive loss of extractable β-endorphin immunoreactivity so that the smaller molecular weight material (β-endorphin) disappeared more rapidly than the higher molecular weight material (β-lipotrophin). Pituitaries from neonates and foetuses were found to contain proportionally more β-endorphin than pituitaries from adults (β-lipotrophin/β-endorphin ratio was 2.76 ± 0.43 in adults, 0.93 ± 0.11 in neonates and 0.47 ± 0.09 in foetuses). β-endorphin-like material from gel filtration was found to separate into two components on SP-Sephadex C-25 cation exchange chromatography. The more basic of these eluted identically with synthetic human β-endorphin in reversed phase HPLC, displaced tracer parallelly both in radioimmunoassay and opiate receptor binding assay and had a receptor binding potency not significantly different from that of synthetic β-endorphin. The less basic component has a smaller molecular weight than β-endorphin and represents a formerly unrecognized human pituitary endorphin-like peptide. The results suggest that β-endorphin is a naturally occurring peptide in human pituitary.