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Isolated rat pancreatic islet extracts were submitted to reverse phase high pressure chromatography (HPLC) and assayed for thyrotrophin releasing factor (TRF), glucagon and insulin in radioimmunoassays (RIA). Our anti-TRF serum was prepared by immunizing rabbits with TRF conjugated to bovine thyroglobulin (bTG) by bisdiazobenzidine. Anti-glucagon and anti-insulin sera were from a commercial source (Novo, Denmark). The concentrations of TRF, glucagon and insulin were 0.05 ± 0.02, 0.06 ± 0.02 and 1.0 ± 0.2 pmol/islet, respectively. Formalin-fixed pancreatic sections were stained for TRF, glucagon and insulin by peroxidase, antiperoxidase (PAP) complex method. When the adjacent sections were stained for glucagon or insulin, it was observed that TRF and glucagon-specific peroxidase reactions were confined to the marginal islet cells, but insulin reactions to the central cells. The TRF-specific peroxidase reaction was clearly reduced when the anti-TRF serum was adsorbed with TRF-TG-Sepharose, whereas it was unchanged when the antiserum was pre-incubated with synthetic TRF. The present HPLC results suggest that the islets contain TRF. Immunocytochemical studies show that the TRF-immunoreactive material, either synthesized or bound, is localized in the marginal islet cells.

The mid-fractions in gel filtration of rat serum proteins were shown to be the most potent in abolishing the biological activity of TRH during incubation in vitro. The kidneys were not able, in vitro or in vivo, to restore the biological activity of inactivated TRH. Biologically active TRH, labelled TRH and serum-inactivated labelled TRH were excreted in the urine in equal amounts.

Iodination of thyrotropin releasing factor (TRF) with chloramine-T and NaI at pH 7.5 produces both mono- and diiodo-TRF as well as unreacted TRF and I − . The four compounds can be separated by ion-exchange chromatography on SP-Sephadex C-25, using a stepwise gradient of 0.01 m MH 4 OAc at pH 3.5, 5.2 and 7.2. The monoiodo-TRF was characterized by nuclear magnetic resonance, mass spectra, and elemental analysis as [Im-5-iodo-His 2 ]-TRF while the diiodo-TRF was similarly characterized as [Im-2,5-diiodo-His 2 ]-TRF. Both iodination products showed no thyrotropin releasing activity at doses up to 1 μg in the in vivo mouse bioassay. The specific binding of mono- and diiodo-TRF was measured against three different TRF antisera. In the absence of TRF, antisera A and C bound more diiodo-TRF than monoiodo-TRF while antiserum B seemed to bind both tracers equally. In all cases steeper curves were produced when diiodo-TRF was displaced by synthetic TRF.

The insulinotropic and glucagon-releasing activity of acid extracts of rat hypothalami were tested in two bioassay systems using short-time incubation of isolated rat pancreatic islets and a rat insulinoma (RIN) cell line. Release of insulin and glucagon into the incubation medium was measured by radioimmunoassay. The major insulin-releasing and glucagon-releasing activity eluted in a broad zone in Sephacryl S-zoo gel filtration in 30% acetic acid corresponding to the molecular weights between approximately 10 and 40 kD. The highest activity was eluted in a zone corresponding to 14 kD and was purified to homogeneity by means of two steps of reversed—phase HPLC. Amino acid analysis and SDS polyacrylamide gel electrophoresis indicated that the biologically active material was the rat small (myelin) basic protein characterized previously by Dunkley & Carnegie (1974). The purified rat hypothalamic material showed insulinotropic and glucagon-releasing activity indistinguishable from that of purified porcine myelin basic protein, and lost its insulinotropic activity when incubated with anti-myelin basic protein immunosorbent. We conclude that the major insulin-releasing and glucagon-releasing activity in acid extracts of the high-molecular-weight fractions of rat hypothalami is myelin basic protein.

Male Sprague-Dawley rats were given graded doses of methylmercaptoimidazole (MMI), propylthiouracil (PTU), KClO4 or KI in drinking water for 4 days, or the lowest effective dose of each drug for various times. The rats were sacrificed at 1–2 p. m. and serum T3, T4 and TSH concentrations were measured by radioimmunoassays. It was found that administration of 5 mg/l of MMI, 10 mg/l of PTU and 100 mg/l of KClO4 for 4–14 days induced a transient rise in serum TSH and a fall in serum T3 or T4 or in both. The effects of KI were not consistent. In another series of experiments, PTU (10 mg/l) was given in drinking water for 4 days, and then graded doses of T3 or T4 were given iv, or 100 ng of TRH was injected into a tail vein, or the animals were exposed to 4°C for 30 min. The initial high TSH levels were further increased by TRH and cold and decreased by T3 and T4. The PTU-treated animals had goitres after 4 days. We infer that low doses, that is to say 10–100 times lower than previously described, of antithyroid drugs induce a hypothyroidism characterized by an increased TSH level and a decreased serum T3 or T4 level or both. A 4 days' treatment with PTU (10 mg/l in tap water) is a suitable tool for studying the effect of various conditions on TSH secretion.

The effects of synthetic GHRH-(1-44) (1 microgram/kg, iv), clonidine (0.15 mg/m2, orally), L-dopa (0.5 g, orally), and insulin (0.1 IU/kg, iv) on plasma immunoreactive (ir) GHRH and GH levels were determined in normal men, aged 31-46 yr (n = 4-8). In addition, plasma ir-GHRH and GH concentrations were determined before and after the administration of clonidine in six younger men, aged 19-25 yr. GHRH was extracted from plasma using Sep-Pak C18 cartridges and measured with a mid-portion-specific GHRH antiserum. The mean plasma ir-GHRH and GH levels ranged from 9-11 ng/L and 0.5-1.5 microgram/L, respectively, in the older men during a 2-h control study. After GHRH administration, the mean plasma ir-GHRH concentration increased to a peak of 512.5 ng/L at 3 min and GH to a peak of 9.2 micrograms/L at 10 min. Clonidine resulted in a significant increase in mean plasma GH levels (P less than 0.05) in the younger men, but not in the older men. Plasma ir-GHRH concentrations did not change after clonidine. L-Dopa increased plasma ir-GHRH at 60 min (P less than 0.05) and GH at 60-120 min (P less than 0.05). Insulin-induced hypoglycemia increased plasma GH levels (to a mean of 23.8 micrograms/L at 60 min; P less than 0.001), whereas plasma ir-GHRH levels did not change. We conclude that the mechanisms of the various GH stimulation tests differ. Some GH responses, including those induced by insulin, do not appear to be mediated by GHRH.

To evaluate the role of vasopressin in the regulation of atrial natriuretic peptide (ANP) secretion, the plasma, atrial, ventricular, and hypothalamic levels of ANP were measured in Long-Evans (LE) and vasopressin-deficient Brattleboro (DI) rats. Total atrial immunoreactive ANP (IR-ANP) as well as right auricular IR-ANP concentration were higher in the DI than in the LE rats, whereas no significant difference was noted in left auricular IR-ANP concentration. In the left ventricle of DI rats, the IR-ANP concentration was 82% greater than that in the LE rats, while no substantial difference was found in the right ventricular IR-ANP concentration between the strains. Normal LE rats had low levels of left ventricular ANP mRNA and barely detectable ANP mRNA in the right ventricle, DI rats showed a 3-fold greater ANP mRNA concentration in the left ventricle than age-matched LE controls, and ANP mRNA levels were also increased in the left auricle of DI rats. The hypothalamic IR-ANP content, but not the concentration, was significantly increased in the DI compared to the LE rats. Despite increased cardiac IR-ANP and ANP mRNA levels, plasma IR-ANP concentrations were similar in the conscious DI rats (97 +/- 9 pg/ml; n = 13) and the LE rats (95 +/- 8 pg/ml; n = 15). Volume expansion (1.1 ml/100 g BW of 0.9% saline, iv) increased right atrial pressure and caused a significant rise in plasma IR-ANP in both strains (P less than 0.01). Elevations of plasma IR-ANP concentrations caused by volume loading were comparable in LE and DI rats in either the absence or presence of exogenous vasopressin (5 ng/kg.min, iv), which, when infused alone, did not significantly influence the plasma IR-ANP concentration. However, the relation between the change in IR-ANP concentration and the change in right atrial pressure shifted to the left, and thus, for a given increase in right atrial pressure, a greater amount of IR-ANP was released in the vasopressin-treated rats than in the control animals. These results demonstrate that although acute volume expansion does not necessarily require endogenous vasopressin for the ANP secretory response, vasopressin increased the ability of volume expansion to induce ANP release, thus modulating the direct mechanical stimulus-induced ANP secretion. The increased left ventricular levels of immunoreactive ANP and augmentation of ANP mRNA levels in Brattleboro rats despite normal left ventricular weight to body weight ratio show that increased ANP gene expression may occur in the ventricles independently of hypertrophy.

The serum and amniotic fluid concentrations of melatonin (MT) were measured by RIA during human labor in different conditions related to the type of delivery and the time of the day of delivery. Serum MT concentrations displayed a normal diurnal rhythm, resembling that of nongravid women; the mean concentration [163.8 +/- 149.6 (+/- SD) pmol/L] at night was significantly (P less than 0.001) higher than that during the day (31.4 +/- 16.3 pmol/L). The amniotic fluid MT concentration, which showed a significant positive correlation to the serum MT concentration (r = 0.625; P less than 0.01), also showed an obvious diurnal rhythm; the mean MT concentration was significantly (P less than 0.01) higher during the night [99.3 +/- 59.3 (+/- SD) pmol/L] than during the day (58.9 +/- 33.5 pmol/L). Reverse phase high pressure liquid chromatography confirmed that the amniotic fluid MT immunoreactivity eluated as synthetic MT. During the night the mean amniotic fluid MT concentration [99.3 +/- 59.3 (+/- SD) pmol/L] was significantly (P less than 0.01) lower than that in serum (178.9 +/- 189.6 pmol/L). The progress of delivery, estimated by cervical dilatation, did not affect serum MT concentrations. Induction of delivery by amniotomy and/or oxytocin, and operative delivery by cesarean section had no effect on serum MT concentrations. Human MT secretion does not seem to be influenced by the physical stress of labor or endocrine changes associated with parturition. The single factor regulating MT secretion during human delivery appears to be the time of day.

The thyroid function of euthyrotic, hypothyrotic and hyperthyrotic rats was studied before and after a 2 weeks' LiCl treatment. 1. 1. The weights of the adenohypophyses, neurohypophyses, adrenals and especially thyroid glands were greater in Li-treated hypo- or hyperthyrotic animals. 2. 2. Serum protein-bound iodine levels were unaffected by Li. 3. 3. The neck 131 I uptake values showed that Li decreased the uptake in euthyrotic animals but increased it in all the other groups. When the mass of the thyroid lobe was taken into consideration, Li increased the uptake in the control and hyperthyrotic animals. 4. 4. In electrophoretic analysis Li caused an increase of thyroidal inorganic iodine in all groups. Chromatographically it was noticed that Li decreased the formation of iodothyronines. The results suggest that Li increases thyroidal iodide, which decreases the formation of T 4 and T 3 . The goitrogenic effect of Li seems to be augmented by a previously pathologic thyroidal state.

After injection of 0.2 mg of TRH iv. concentrations of TRH were estimated in plasma, urine and saliva by T3-TRH bioassay in 5 euthyroid men. Plasma TRH was detectable (over 2 ng/ml) for 15 min and had a half-time of 5 min within the first 5 minutes, Urine TRH level was maximal at 15 min and was about 500-fold that in plasma. Saliva TRH level at 15 min was below 3 ng/ml. The present results are in agreement with those carried out with experimental animals and with 125I-TRH, The rapid appearance of TRH in the urine in bioassayable amounts is evidently due to the inability of the kidneys to reabsorb this hormone. It has previously been reported that injected TRH can be recovered in the urine of the rat in biologically active form (1). In this laboratory it has been shown that 125I-TRH behaves similarly in mice (2). This report deals with studies on TRH elimination in man.

Leppaluoto, J. Electrical stimulation of human thalamic and amygdaloid ana and plasma Cortisol concentration. Acta physiol. scand. 1973. 89. 187–191. The concentration of plasma Cortisol was measured from blood samples drawn through indwelled catheters at 3–5 min intervals in 4 resting healthy subjects and in 5 patients after electrical stimulation of the thalamus and amygdala via chronic electrodes implanted for the treatment of psychomotor seizures. In the healthy subjects (laboratory staft) the plasma Cortisol level, measured by a fluorometric method, was initially high (10 - 37 μg/100 ml), evidently due to emotional affectivity of the experimental situation, and decreased within 10 min by 10μg in 2 subjects and the increased within 10 min by 5–20 μg in 3 subjects. These rapid changes of the plasma Cortisol level, bursts, were significant and are a new physiological phenomenon. In the patients the thalamus and amygdala were stimulated 30 s (pulse duration 1 ms, repetetive frequency 50 Hz, unidirectional square wave pulses) to the voltage level at which the patient felt a subjective sensation (4 -12 V). After repetctive stimulations of thalamus (3 patients, 6 times) and amygdala (2 patients, 3 times) the changes in the plasma Cortisol levels were inconsistent and similar to those seen in the healthy subjects. No conclusion about the function of the pituitary-adrenal axis and amygdala of thalamus was drawn, because the plasma Cortisol values were evidently influenced by their temporal relation to the secretion bursts presented here.

Laisi, U., S. Leisti, J. Leppaluoto, J. Lumio, J. Partanenand P. Virkkunen. Growth hormone and Cortisol secretion in man. Acta physiol. scand. 1973. 89. 192–197. In 4 healthy resting males blood was collected through indwelled catheters at 1–3 min intervals and EEG recorded in a sound-shielded anechoic environment for 30 min (expt. 1). The iminunorcactive plasma growth hormone was at high concentration (10–33 ng/ml) and was secreted in 3 significant bursts in one subject, but was low (0–4 ng/ml) in 3 other subjects. The plasma Cortisol, measured by a fluorometric method, was at low and constant level (mean ±S.D. = 8.9±1.5 μg/100 ml). The EEG vigilance fluctuated between wakefulness and real sleep. The plasma growth hormone concentration tended to be high at wakeful stages and low at sleep stages. There was no correlation between vigilance and plasma Cortisol level. In the middle of the following experiment (expt. 2) a painful faradic current was applied to the forearm for 10 s and blood was collected as in expt. 1. The plasma Cortisol level before the painful stimulus was significantly increased (12.3±4.6 /yg/100 nil) and did not change after the stimulus, in spite of the clear increase of vigilance. The high prc-stimulus level of plasma Cortisol is evidently due to anticipatory mechanisms. We conclude that growth hormone is secreted in bursts which may last as short as 5 min and that the increase of vigilance could trigger the secretion of growth hormone but not that of Cortisol.

In the first experiment Sprague-Dawley rats were given antithyroid drugs i. e. perchlorate (KClO4), carbimazole (CI) and potassium iodide (KI) for 4 weeks, either alone or in combination with lithium (Li) (5 mEq./l) for the last 2 weeks. In combination with CI or KI, Li aggravated the goitre and decreased the synthesis of T3 + T4, as measured by chromatography. The weights of the adenohypophyses increased and those of the adrenals decreased in all the experimental groups. In the second experiment, Li (10 mEq./l) was administered to euthyroid rats for 4, 10 or 20 weeks. The metabolic effects of Li reached a peak at 4 weeks, giving a decreased T3 + T4, a decreased plasma PBI level and an increased plasma bioassayable (McKenzie) TSH level. A definite goitre did not develop until 20 weeks. In the third experiment the disappearance of thyroidal 131I was shown to be delayed in rats treated for 2 weeks with Li or T4 but was accelerated in those rats receiving CI. It is concluded that 1) Li accentuates the antithyroid and goitrogenic potency of antithyroid drugs, 2) Li itself is a mild antithyroid and goitrogenic agent, which exerts its effect by inhibiting the coupling reaction of iodotyrosines and 3) that Li also retards the secretion of thyroid hormones from the thyroid gland.

The salient sign of acutely lethal 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) intoxication in rats is hypophagia along with a prominent body weight loss. Endogenous opioid peptides have been implicated as modulators of food intake. In the present study, female rats of both the most TCDD-susceptible (Long-Evans [L-E]; LD50 9.8 micrograms/kg) and the most TCDD-resistant strain (Han/Wistar [H/W]; LD507200 micrograms/kg) were exposed to a single dose of 50 micrograms/kg TCDD ip. This treatment is usually lethal within 1-6 weeks to all L-E rats and nonlethal to all H/W rats. The animals were killed at 1, 4 or 10 days after the treatment. beta-Endorphin-like immunoreactivity (beta-END-LI) was determined by a validated RIA method in the hypothalamus, pituitary, pancreas, duodenum and plasma. TCDD decreased plasma beta-END-LI concentration by 24-37% at every time point of measurement in L-E rats alone. By contrast, feed-restricted controls exhibited an increase of similar magnitude on day 4. Pancreatic beta-END-LI was also elevated in feed-restricted controls at that time point as compared with either the ad libitum control or TCDD group. TCDD appeared to shrink the pituitary gland in both strains by day 4. Pituitary weight was similarly lowered in TCDD-treated rats and feed-restricted controls at the last time point and this reduction was reflected in pituitary beta-END-LI content. Thus, TCDD affects selectively plasma beta-END-LI levels and this impact correlates with its lethality in these strains.

Background: Assays for endogenous ouabain, a cardiac glycoside believed to be involved in blood pressure and volume regulation, are characterized by laboratory-specific plasma values that are measured by different assays. Because of this variability, our study focused on the development of a new 125I-labeled ouabain derivative for RIA of high sensitivity. Methods: We generated rabbit antisera against a ouabain-thyroglobulin conjugate. A tyrosylated ouabain derivative for radioiodination was synthesized using periodate and sodium cyanoborohydride reagents. Results: Mass spectrometric analyses showed that the main product of the tyrosylating reaction was tyrosyl-ouabain (molecular mass, 702 Da). This was radioiodinated with Chloramine-T and used as a tracer in a RIA, which gave an assay detection limit of 5 pmol/L (4 ng/L), 2–100 times lower than that in the corresponding 3H-RIAs and 2–20 times lower than ouabain ELISAs, making it possible to measure low plasma concentrations of immunoreactive ouabain. Different amounts of SepPak C18-extracted plasma samples displaced the 125I-labeled tyrosyl-ouabain tracer at the same rate at which authentic ouabain was displaced. Plasma immunoreactive ouabain coeluted with authentic ouabain in two different HPLC conditions. Using the new RIA, we found plasma ouabain concentrations, assayed as immunoreactive equivalents, of 10.0 ± 1.3 pmol/L in healthy women and 12.0 ± 0.9 pmol/L in healthy men (mean ± SE; n = 10), as well as 41.2 ± 9.6 pmol/L in rats. The concentrations were 2–90 times lower than those previously reported using different assay methods. Conclusions: Our ouabain 125I-RIA enables reliable measurements of low endogenous concentrations of a ouabain-like compound for both physiological and clinical purposes.

High plasma immunoreactive Pendorphin levels have been observed in the human fetus at delivery (1, 3) and a correlation between high &endorphin and low pH in arterial cord plasma has been reported (3). We have measured umbilical cord plasma levels of immunoreactive P-endorphin in normal vaginal births and compared those with P-endorphin levels in breech presentation and vacuum extractor deliveries.

Genus Rangifer populates the northernmost habitat of the Cervidae and shows alterations in body weight, skeletal growth rate and nutritional state with the marked seasonal changes in its environmental conditions. Body weight and measurements have long been the standard criteria for evaluation of growth in domestic animals. Body weight has been also used most by workers to describe growth and body size in reindeer and caribou. The numerical values of the weight to length ratios of skeletal bones are not so much expressions of growth rates, but rather of the growth patterns of these bones. Body measurements and ratios are useful taxonomic criteria and are used in Rangifer systematics. The results indicate that body ratios, such as shoulder height/foreleg length and shoulder height/back length are independent of body size and thus primarily of genetic origin (Nieminen & Helle 1980). The objective of this study was to measure the apparent ageand sex-related growth and the seasonal changes in body weight and body measurement in semidomesticated reindeer. During different seasons in 1969 85 the body weight was measured in Kaamanen experimental station and in different herding cooperatives from 2932 reindeer females ranging from birth to 14 years of age and from 1037 males ranging from birth to 10 years of age. General physical development was measured after the first 4 months of life by chest circumference and back length taken from 1490 females and 510 males. The mean birth-weight of female calves was 5.0 kg. Male calves weighed 0.3 kg more. After the first 6 months of life, body weight of reindeer calves followed alternating periods of positive and negative growth. The body weight increased until the age of about 3.5 years in females and until the age of 5 7 years in males. In winter both young and adult females lost between 13 and 19% of their prime autumn weight. However, the percentage of prime autumn weight lost by males in winter increased from about 10% the first year to 33% at maturity. The fluctuations of body weight are largely attributed to the seasonal depostition and mobilization of fat reserves. Growth of reindeer ceased in winter because of either an inherent physiological rhythm or a decline in nutritive value of the diet, or both. The body weight of female reindeer was highly correlated with the back length (r=0.809) and chest circumference (r=0.860). Similar relationships were also found for male reindeer (r= 0.892 and 0.872, respectively). However, the best relationships were noted between the body weight and the joint measure (back length + chest circumference) in female (r=0.877) and male reindeer (r=0.941). Similar relationships have been shown for domestic species. There were some differences in relationships between the body weight and the joint measure in young and over 3-year-old females and males. Differences were also found in these relationships during autumn and winter.

Availability of radioimmunoassay for growth hormone releasing hormone (GHRH) in plasma has made it possible to measure circulating GHRH concentrations during stimulation of growth hormone (GH) secretion. It has been previously shown that administration of L-dopa increases plasma levels of GHRH, followed by an elevation of plasma GH concentration. Ornithine or clonidine do not seem to affect plasma levels of CHRH. To elucidate the role of GHRH in the stimulation of GH secretion during pharmacological stimulation tests we measured plasma immunoreactive GHRH and GH levels in 8 healthy adult men during insuline induced hypoglycemia (IH) and after oral administration of clonidine of L-dopa. Plasma samples for the determination of GHRH levels were purified with octa-decyl-silica cartridges and assayed by a radioimmunoassay specific for the midportion of human GHRH. Mean GH response (SEM) after administration of L-dopa was 8.67 ug/l (2.9), 2.22ug/l (1.0) in clonidine test and 24.33 ug/l (4.4) during insuline induced hypoglycemia. Mean GHRH responses rospectively were 9.64 ng/l (2.6), 6.65 ng/l (2.3) and 4.25 ng/l (3.4). The plasma GHRH peaks preceded or coincided with GH peaks in all subjects after L-dopa administration. In the clonidine test and IH no correlation was found between the GHRH and GH peaks. These results confirm that the effect of L-dopa on GH secretion is mediated by GHRH, whereas other factors than GHRH seem to play a major role in GH stimulation by IH and clonidine test.

Obese children have in a serum fraction of m. wt. 1,000-5,000 an insulin-releasing activity (IRA) occurring also in some normal-weight children, particularly in infants (Lautala, P.: Acta Univ. Oul D 32 Med. Interna Paediatr. 3:1978). The aim of the present study was to further characterize the IRA. The serum fractions of m. wt. 1,000-5,000 were prepared by ureg treatment and filtration of the sera through four Amicon-membranes of different porus sizes. Eight serum fractions containing IRA from four obese and four non-obese children were subfractionated with reversed-phase high pressure liquid chromatography into four subfractions (SF). IRA of the fractions and their SFs was measured by perifusion of isolated rat islets. The first SF did not in any case contain IRA. The second SF was positive in all eight children. The third SF contained IRA in six children, while the fourth SF was positive in four children. IRA did not show a relationship to the radioimmunologically measured concentrations of pancreatic glucagon, gut GLI or GIP in the serum fractions. These results indicate that serum fractions of m. wt. 1,000-5,000 from obese and some non-obese children contain at least one insulin-releasing substance, which is obviously not identical with glucagon or GIP.

The authors wished to test the extent to which TSH, ACTH or both, are secreted by pituitary cells during the stress of acute exposure to cold. Differences in secretion were found between rat and rabbit.