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Background: High circulating concentrations of N-terminal fragments of A- and B-type natriuretic peptides (NT-proANP and NT-proBNP) identify patients with impaired cardiac function. ProANP-derived peptides are particularly sensitive to increased preload of the heart and proBNP-derived peptides to increased afterload; therefore, combining the information from the ANP and BNP systems into a single analyte could produce an assay with increased diagnostic and prognostic power. Methods: We prepared a hybrid peptide containing peptide sequences from both NT-proBNP and NT-proANP (referred to as NT-proXNP) by recombinant techniques and used it to develop a RIA combining weighed concentrations of NT-proANP and NT-proBNP into a new virtual analyte, NT-proXNP. We used the novel method to measure the circulating concentrations in healthy persons and in patients with cardiac disorders. We also characterized the assay by HPLC analysis of the immunoreactive molecular forms in human plasma and serum. Results: The results from the novel assay correlated well with independent home-made NT-proANP and NT-proBNP assays (r2 = 0.75–0.85) as well with the arithmetic sum of NT-proANP and NT-proBNP (r2 = 0.92). Patients with valvular heart disease (VHD) and coronary artery disease (CAD) had significantly increased NT-proXNP concentrations. The areas under the curve (AUC) of the NT-proXNP assay in detecting VHD and CAD (0.961 and 0.924, respectively) were significantly larger than the AUC of either NT-proANP (0.947 and 0.872) or NT-proBNP (0.913 and 0.782) assays. HPLC analysis showed that the novel NT-proXNP assay detects two major classes of circulating immunoreactivity corresponding to peptides derived from NT-proANP and NT-proBNP. Conclusions: Our novel immunoassay mimics the physiologic signaling system working in the body by converging the information obtained from the activation of ANP and BNP into a single virtual analyte, NT-proXNP. It appears to have a diagnostic efficiency equal to or slightly better than that of individual NT-proANP or NT-proBNP assays.

Background: The N-terminal fragments of A- and B-type natriuretic peptides (NT-proANP and NT-proBNP) are powerful markers of cardiac function. The current assays require refinement with regard to standardization with native calibrators and the ability to detect the actual circulating forms.Methods: The following peptides were prepared with recombinant methods: NT-proANP, NT-proBNP, proBNP1–108, and Tyr0-proBNP77–108. Fifteen peptides of 13–22 amino acids, spanning the sequences of NT-proANP and NT-proBNP, were prepared by solid-phase peptide synthesis. Two immunoassays for NT-proANP and four for NT-proBNP were set up, each with a different epitope specificity. The assays were applied for the measurement of NT-proANP and NT-proBNP in healthy individuals and in patients with acute myocardial infarction. The circulating molecular forms were analyzed by gel-filtration and reversed-phase HPLC.Results: According to the HPLC analyses, circulating NT-proANP consists mainly of the full-length peptide, with some degradation at both ends. In contrast, circulating NT-proBNP is very heterogeneous. Most immunoreactive NT-proBNP is significantly smaller in size than NT-proBNP1–76, with truncation at both termini. The smallest fragments can be detected by assays directed at the central part of NT-proBNP only; assays directed at the ends gave 30–40% lower values. Despite the difference, the various assays correlated reasonably well with each other (r2 = 0.77–0.85). In patients with acute myocardial infarction, NT-proANP and NT-proBNP concentrations were 1.8–2.3 and 4.2–4.5 times higher than in healthy individuals. The development of heart failure further increased the concentrations.Conclusions: Molecular heterogeneity of the circulating forms causes a serious risk of preanalytical errors in assays for NT-proBNP and, to a lesser extent, NT-proANP. The development of a sandwich assay for NT-proBNP would be especially challenging. The most robust and reliable assays use antibodies directed at the central portions of NT-proANP or NT-proBNP.

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

The reindeer ( Rangifer tarandus tarandus ) is a semidomesticated holarctic ruminant subject to economic activity. Our research was carried out in Northern Finland, at latitude 69°10'N, where the daily light-dark rhythm has a polar pattern: constant darkness starts on November 26th and lasts about 10 weeks, while the polar day of about 10 weeks starts on May 15th. We had previously found distinct daily rhythms in reindeer melatonin secretion in autumn, winter and spring but not at all in summer. Here we determine how the daily melatonin pattern develops after the period of polar day and how it disappears at the beginning of that period. Melatonin showed increased concentrations very soon after the first sunset. Thereafter the daily pattern developed gradually and was fully developed around the autumn equinox. Its shape was asymmetric and the maximum amplitude occurred near the end of the dark period. The disappearance of this daily pattern in spring was also gradual. The maximal concentrations depended on ambient illumination, especially the duration of the dark period. In spring the reindeer appeared to be more sensitive to light than in autumn. Additionally illumination of the same magnitude suppressed serum melatonin levels more effectively before midnight than afterwards, which is in accordance with the asymmetric secretion pattern. Melatonin secretion was always suppressed when the ambient illumination exceeded 1,000 1x. The pattern of melatonin secretion in reindeer is different from those described earlier and hence the reindeer may serve as a model for melatonin secretion at high latitudes. We suggest that, in addition to the duration of the melatonin secretion, changes in amplitude are also of importance in the reindeer.

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 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.

Acute effects of lithium chloride (LiCl, 5 mEq./kg × 3), rubidium chloride (RbCl, 5mEq./kg×3), carbimazole (0.05 mEq./kg × 3) and short-term transfer stress on thyroid iodine metabolism were studied in rats fed a normal-iodine diet (NID). In addition, the effect of graded doses of LiCl (0–7.5 mEq./kg × 3) was studied in rats fed a low-iodine diet (LID). In the animals on NID carbimazole, LiCl and RbCl decreased the 4 h uptake of 131I. In chromatography it was found that only carbimazole and LiCl significantly decreased the synthesis of T3 + T4. Plasma TSH (radioimmunoassay) levels were slightly decreased by short-term transfer stress, RbCl and LiCl, but increased by carbimazole. In the animals on LID, the 131I uptake at 4 h decreased and correspondingly the plasma radioactivity increased with increasing doses and plasma concentrations of Li+. In chromatography the relative amounts of iodothyronines and DIT decreased, that of MIT increased, and the percentage of iodide remained unchanged. With a minimal effective dose, Li reduced only T4 and T3, with the higher dose DIT, then iodide and the total uptake of 131I, and lastly MIT. The plasma corticosterone level tended to increase and TSH level to decrease as the plasma Li concentration increased. Thus lithium in massive doses seems to be an antithyroid agent, its effects on thyroidal uptake and distribution of 131I resembling that of very small doses of propylthiouracil. The inhibitory effects of Li are dose-dependent. The decrease in plasma thyroxine level probably initiates the series of events which may lead to hypothyroidism, goitre or both conditions.

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.

Seven patients suffering from severe endometriosis were treated with danazol 200 mg × 3 daily for 6 months. Clinical symptoms were alleviated and menses disappeared in response to the treatment. After cessation of the treatment the menstrual bleedings returned in 1–3 months. Blood samples for determination of gonadotrophins, prolactin (Prl), oestradiol (E2), progesterone, thyroid hormones and thyrotrophin in radioimmunoassays were taken and a combined TRF and LRF test carried out in the follicular phase before treatment, at the 6th month of treatment and after reappearance of the first menses. There were no statistically significant changes in the basal levels of serum FSH, LH or TSH during the danazol treatment. Neither was there any change in episodic secretions of FSH, LH or Prl, as determined by the mean coefficients of variation of the hormone levels in seven consecutive samples taken at 20 min intervals. On the other hand, serum E2, Prl and thyroid hormone levels were significantly decreased in the 6th month of treatment. In the TRF-LRF test the responses of serum FSH and LH were significantly higher and those of serum Prl and TSH significantly lower during danazol treatment than before. Prl responses remained lowered after the treatment. It appears that low serum oestrogen levels, induced by the danazol treatment, sensitize the pituitary gonadotrophs to exogenous LRF, but make the sensitivity of thyrotrophs and lactotrophs lower to exogenous TRF. These results thus indicate that danazol does not make the pituitary gonadotrophs insensitive to LRF, but danazol may rather inhibit the secretion of hypothalamic LRF.

In this method for rapid separation of beta-endorphin (beta-EP) and beta-lipotropin (beta-LPH) in plasma, most of the plasma proteins in a 0.5- to 2.0-mL sample of plasma are precipitated with acetonitrile at pH 4.7. beta-EP and beta-LPH in the supernate are completely separated by liquid chromatography on a cation-exchange column, with gradient elution with volatile buffers, and are eluted in 1.5- to 2.0-mL volumes. After lyophilization, the redissolved beta-EP and beta-LPH are determined by radioimmunoassay. The respective mean concentrations of immunoreactive beta-EP and beta-LPH in plasma of nonpregnant women were 4.0 (SD 2.0) and 5.1 (SD 3.0) pmol/L; during labor, 33.3 (SD 25) and 69.6 (SD 41) pmol/L; and in venous plasma from umbilical cords after spontaneous labor, 38.6 (SD 18) and 38.7 (SD 19) pmol/L.