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
Anna-Stina Suhonen, Juhani Leppäluoto and Jorma Salmi
Abstract. Urine samples from 8 healthy subjects, from 16 patients with primary hypothyroidism and 8 patients with Graves' hyperthyroidism were pre-purified in SP-Sephadex-C-25 cation-exchange-chromatography, subjected to reverse phase high-pressure liquid chromatography (HPLC) with 0.01 M ammonium acetate pH 4 as a polar and propanol as a non-polar solvent with a 1%/min gradient and assayed in our TRH radioimmunoassay. Urine TRH-immunoreactivity levels were measured before and after 3 months of treatment with thyroxine or methimazole. The urine TRH-levels in healthy subjects were 5.5 ± 1.4 ng/l (mean ± sem, n = 8).
In the hypothyroid patients, the urine TRH levels were 50.6 ±40 ng/l before and 71.7 ± 45.3 ng/l after 3 months of treatment with thyroxine. These values did not significantly differ from those in healthy subjects. The large variations were due to highly elevated values in 3 patients. In 2 hypothyroid patients with initially high urine TRH values, 67 and 657 ng/l, urine TRH was measured 5 and 18 months later and was found to have decreased to 5 and 11 ng/l. In the hyperthyroid patients, urine TRH levels were 10.3 ± 3.9 ng/l before and 8.9 ± 3.3 ng/l after the treatment with methimazole and did not differ significantly from the levels in healthy subjects. After 3 months of treatment, the hyper- and the hypothyroid patients were euthyroid.
Our results show, that, except in 2 hypothyroid patients, there does not appear to be any relationship between urine TRH levels and serum TSH or thyroid hormone levels in hypothyroid and hyperthyroid patients.
Olli Vakkuri, Aarre Kivelä, Juhani Leppäluoto, Maija Valtonen and Antti Kauppila
Vakkuri O, Kivelä A, Leppäluoto J, Valtonen M, Kauppila A. Decrease in melatonin precedes folliclestimulating hormone increase during perimenopause. Eur J Endocrinol 1996;135:188–92. ISSN 0804–4643
Melatonin, the hormone of the pineal gland, which in animal studies has been found to inhibit aging processes, is secreted in smaller amounts towards senescence. Menopause, an aging process in women, is known to be associated with typical changes in gonadotropin and sex steroid secretion. Our main objective was to study the possible role of melatonin in the hormonal regulation of menopause. This study focused on detailed changes in melatonin and follicle-stimulating hormone (FSH) secretion cross-sectionally in pre- to postmenopausal females. Special attention was paid to females aged around 50 years, which is the mean menopausal age. Seventy-seven healthy female volunteers aged 30–75 years were the subjects of this study. Melatonin was measured radioimmunologically from nocturnal urine collected between 20.00 and 08.00 h, and FSH and melatonin from blood samples taken at 09.00 h. Nocturnal urinary excretion of melatonin was found to decline significantly from premenopause to postmenopause. The youngest premenopausal women (age group 30–39 years) excreted the highest amounts of melatonin (21.1 ± 2.2 pmol/h, mean±sem, N = 17). In the age group 40–44 years the excretion declined by 41% (p < 0.05). The second significant decline (35%, p < 0.05) took place between the age groups 50–54 years and 55–59 years. A declining trend as a function of age was also seen in morning serum melatonin. Serum FSH rose sharply to high levels before the age of 50 (p < 0.01) and remained at a high level thereafter. Urinary melatonin correlated negatively with serum FSH (r = −0.32. p < 0.05). In conclusion, the inverse changes in melatonin and FSH secretion during the perimenopausal years, with the sharpest decline in nocturnal excretion of melatonin far before menopause, suggest that melatonin may be permissively linked to the initiation of menopause.
O Vakkuri. Department of Physiology. University of Oulu, Kajaanintie 52 A, 90220 Oulu, Finland
Hannu Martikainen, Juha Tapanainen, Olli Vakkuri, Juhani Leppäluoto and Ilpo Huhtaniemi
Abstract. This study was aimed at elucidating the possible effects of a large annual variation in photoperiodicity on the secretory activities of the pineal gland, pituitary and testes. Serum daytime melatonin, FSH, LH, prolactin (Prl), testosterone and oestradiol concentrations were determined monthly over a year in 24 healthy young adult men (except for melatonin which was analysed only in 11 subjects) in northern Finland, where the day length is 22 h in mid-summer and 3.5 h in midwinter.
Serum daytime melatonin levels showed two annual peak values, in December and May, and a nadir was observed in August. The absolute values of the other hormones measured did not show significant month to month variation over the observation period. When hormone levels were calculated as percentages of the individual annual means, several significant differences were found between monthly levels. The melatonin peak in May (133 ±20%, se, of the annual mean) was associated with significant increases in LH (110 ±4%) and FSH (107 ± 3%). Prl levels (115 ± 9%) reached a maximum in January. The nadirs of melatonin and the pituitary hormones measured were seen in August. Oestradiol showed the highest values in April-June, but no significant variation was found in serum testosterone levels. Positive correlations were observed between FSH and LH (r = 0.41, P <0.01), and Prl and LH (r = 0.26, P< 0.01), whereas Prl and testosterone (r = −0.17, P< 0.01) were inversely correlated.
This study indicates circannual rhythmicity of peripheral serum daytime melatonin, gonadotrophin, Prl and oestradiol levels, but this variation was not related to extremes in daylight and therefore seasonal factors other than light may regulate this circannual variation.
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.