Harvey J. Guyda

Studies of prolactin secretion in human pregnancy

Recent studies have confirmed the presence of a separate (HPr) human prolactin molecule. Measurement of HPr concentrations has been performed in normal and abnormal gestation and during the menstrual cycle. HPr rises throughout gestation with a return to prepregnant concentrations by the 7th postpartum day. Variable increases in plasma prolactin were observed after intravenous administration of arginine, especially after the 30th week of gestation. Puerperally, prolactin rises in response to sucking. Amniotic fluid prolactin levels are 100 times those of maternal or fetal blood. No significant change in basal HPr is seen during the menstrual cycle. The ability to measure this new pituitary polypeptide by radioimmunoassay permits investigation of its role in human gestation.

Preliminary report on a mass screening program for neonatal hypothyroidism

We have recently developed an immunoassay that can measure thyroxine rapidly and accurately in the eluate of 40 mul of dried blood spotted on filter paper at the fifth day of life. The method is completely automated and by using the samples received by the Central Laboratory of the Quebec Network for Genetic Medicine and their follow-up facilities, we are now screening every newborn in the province of Quebec for neonatal hypothyroidism. To date, from 47,000 measurements, three newborn infants with abnormally low TBG and seven hypothyroid infants have been detected. From these data we conclude that the frequency of congenital hypothyroidism is about one in 7,000 births and that our method is effective in detecting thyroid hormone abnormalities with an acceptable percentage of false positive measurements; no false negative results have occurred to our knowledge.

Peroxide(s) of vanadium: a novel and potent insulin-mimetic agent which activates the insulin receptor kinase.

The actions of insulin, vanadate (V) and hydrogen peroxide (H2O2) on IGF-II binding and insulin receptor tyrosine kinase activity were studied in rat adipocytes. Incubating adipocytes with a combination of V plus H2O2 resulted in a potent synergistic effect on both the increase in IGF-II binding and the activation of the insulin receptor kinase. Catalase, which removes H2O2, abolished this synergism if added at the time of mixing of V plus H2O2 but not if added 10 min. later, suggesting that the formation of peroxide(s) of vanadate generated a potent insulin mimicker. The data support a critical role for the insulin receptor kinase in insulin action. The novel insulin-mimetic compound, a presumed peroxide of vanadate, could prove useful for investigating insulin action and may be valuable for treating insulin resistance.

Acute Reversal of the Enhanced Insulin Action in Trained Athletes: Association with Insulin Receptor Changes

We studied the effect of aerobic training and detraining on insulin-stimulated glucose disposal and on erythrocyte insulin receptor binding. Seven endurancetrained athletes were studied at 12 h, 60 h, and 7 days after cessation of training and compared with three untrained, age- and weight-matched controls. The metabolic clearance rate of glucose as measured by the euglycemic clamp technique was 15.6 ± 1 . 8 ml/kg/min (mean ± SEM) in the trained subjects 12 h after the last bout of exercise compared with 7.8 ± 1.2 ml/kg/min in the untrained control group. When the trained subjects refrained from physical training, the metabolic clearance rate decreased to 10.1 ± 1 . 0 ml/kg/min at 60 h and further to 8.5 ± 0.5 ml/kg/min after 7 days of detraining. The percentage of specific insulin binding to young erythrocytes (density 1.089–1.092), isolated by density gradient centrifugation, decreased from 10.4 ± 0.9 at 12 h after the last exercise to 8.1 ± 0.7%/3 × 109 cells after 60 h of detraining (P < 0.001). The decrease in insulin binding to erythrocytes was almost entirely accounted for by a decrease in the number of insulin receptors. We conclude that the increase in peripheral insulin action seen in trained athletes is rapidly reversed, possibly by a mechanism separate from other phenomena associated with chronic training. The parallel findings of decreased in vivo insulin action and decreased insulin binding in young erythrocytes suggest that modulation of in vivo insulin response by detraining may be at least partially mediated by changes in insulin receptor number. Isolated young erythrocytes are better indicators of acute insulin receptor modulation than are whole cell preparations.

Purification of Human Prolactin

Publisher Summary The existence of the pituitary hormone prolaetin in primates has been the subject of debate for many years. Although numerous clinical and experimental observations suggested the existence of this hormone in man, its isolation had proved to be very elusive; preparations from human pituitary glands having lactogenic activity were invariably contaminated by growth hormone, raising the possibility that perhaps in man growth hormone functions also as the lactogenic hormone. It is only in the past 2 years that the chemical isolation of prolactin from human pituitary glands has been achieved, establishing unequivocally that prolactin exists in man and is distinct from growth hormone. Chemical and biological studies indicate that human prolactin purified by methods currently in use is essentially homogeneous and biologically active. The methods outlined for the purification of human pituitary prolactin are very reproducible with respect to the yield and purity of the final product. The yield is about 50 μg prolactin per gland. The purity is adequate for chemical studies. Full biologic activity is retained. Amniotic fluid appears to be a promising alternative source of human prolactin.

Serum prolactin levels in humans from birth to adult life.

Extract: Serum prolactin (HPr) and serum growth hormone (HGH) were determined by double antibody radioimmunoassay methods. Markedly elevated levels of serum prolactin with considerable variation were observed in the neonatal period. No significant difference was observed in six matched arteriovenous cord blood samples. No sex difference was noted in the full term infants, whereas the mean value for 24 premature male infants in the 1st week of life (190 ± 17 ng/ml sem) was significantly higher (P < 0.001) than mean values for 34 premature female infants (104 ± 10 ng/ml sem). During the first year of life, the mean prolactin value for both boys and girls was approximately 10 ng ml. Mean prolactin levels for both male and female children, aged 2–12 years, were approximately 5 ng/ml. Mean levels for the adolescent female were not increased significantly over those for adolescent males. However, the mean prolactin level of all values determined for adult females (7.9 ± 0.40 ng/ml sem) was significantly increased (P < 0.001) over the mean level for adult males (5.2 ± 0.55 ng/ml, sem). Daily serum prolactin throughout the menstrual cycle in six normal female subjects was compared with daily serum HLH levels. Considerable fluctuation was evident, particularly in the luteal phase, where the mean prolactin level was observed to be statistically higher (P < 0.005) than the mean follicular phase level.Insulin hypoglycemia did not produce a significant increase in serum prolactin in 10 normal subjects, whereas arginine infusion produced a twofold increase in mean serum prolactin at 30 min with a return to basal values by 60 min. Glucagon administered intravenously did not produce any significant change in the already elevated levels of prolactin observed on days 1 and 3 of life. Serum prolactin was uniformly and completely suppressed by L-dopa in six subjects for 1–4 hr following a single oral dose of 250 mg. In normal children, the maximal increases in both prolactin and thyroid-stimulating hormone (TSH) were observed at 15–30 min after the intravenous injection of thyrotropin-releasing hormone (TRH) and values were still slightly elevated at 120 min after injection.These studies document the pattern of secretion of serm prolactin from birth to adulthood. The physiologic basis for the increased levels of serum prolactin in the neonate has not been clarified by our studies. Significantly increased levels of prolactin are observed at a time when maximum increases in neonatal breast hypertrophy are observed clinically. Significant mean differences are observed in the adult female population compared with adult males. L-Dopa acts at the hypothalamic level to alter pituitary secretion via alterations in releasing and inhibiting hypothalamic hormones, whereas TRH acts directly on the pituitary. Our studies indicate that responses to these agents in the prepubertal child are qualitatively similar to those in adults. The response of the neonatal hypothalamic pituitary axis to these agents remains to be studied.Speculation: These studies provide a basis for interpretation of disturbances in hypothalamicpituitary regulation of prolactin secretion. Additional studies are required to define the physiologic roles for prolactin in the human during both intrauterine and extrauterine life as well as the normal ontogeny of control mechanisms for prolactin secretion in the neonate and infant. The availability of sufficient purified human prolactin for metabolic balance studies will also be required to enhance our knowledge of this recently isolated human hormone.

Failure of naloxone to influence physiological growth hormone and prolactin secretion.

Morphine and the opioid peptides, fl-endorphin, [MetZ]enkephalin and [LeuZ]enkephalin are reported to stimulate growth hormone (GH) and prolactin (PRL) secretion in the rat2-5,7, 9,14. Hormone release has been demonstrated after both systemic [intravenous (IV), intraperitoneal (IP) or subcutaneous (SC)] and intracerebral ventricular (ICV) injections; the amounts given have, in general, been large (1-100 mg systemic or 1-100 #g ICV) and anesthetized rats have commonly been used. Shaar et al. is reported that enkephalin analogs administered SC in doses of 10 mg/kg also elicited GH and PRL release in the female rat. The site and mechanism of action of these effects has not been determined. A hypothalamic or other brain locus is suggested by the fact that the substances fail to elicit hormone release from pituitary glands in vitro 1. Martin et al. 1° showed that large hypothalamic lesions failed to completely block morphine-induced GH release and postulated that the effects were mediated at the level of the median eminence. Both enkephalin and fl-endorphin containing cell perikarya occur in the hypothalamus with axons that terminate in the median eminence ~,s. The role of endogenous opioids in the regulation of physiologic GH and PRL secretion is controversial. Previous reports in which single samples of blood were obtained by decapitation suggested a suppression of baseline G H and PRL levels after administration of naloxone, an opioid antagonist 2,7. Stress and suckling-induced PRL responses are also reported to be inhibited by prior administration of opioid antagonists 7. The objective of the present studies was to investigate the effects of naloxone on GH and PRL secretion in unanesthetized rats and in human subjects during sleep. Experiments in our laboratory have documented that physiologic GH secretion in unanesthetized, freely behaving rats is episodic with abrupt surges of secretion occurring at intervals of 3-4 h12, ~9. Basal PRL secretion in such rats is low (less than 10 ng/ml) with infrequent small secretory surges. Sleep-associated changes in GH and PRL secretion are well documented in man, a surge of GH occurring within 2 h of sleep

Short term tissue culture of human midterm and term placenta: Parameters of hormonogenesis

Abstract Monolayer cultures of human midterm and term placentae have been established following trypsin dispersion of placental minces. Maintenance of endocrine function was monitored by the concentrations of specific hormones in the culture media. At either gestational age the cultures 1) secrete estradiol-17β(1) and estrone (in a ratio of about 1: 20) and aromatize 3H- or 14C-dehydroepiandrosterone sulfate and 14C-androstenedione, estrogen production being markedly enhanced by addition of dehydroepiandrosterone (10−6M) to the culture medium; 2) metabolize 3H-pregnenolone to progesterone and 14C-cortisol to cortisone; and 3) produce increasing amounts of chorionic gonadotropin and decreasing amounts of placental lactogen during the first week in culture. It is proposed that the model is highly suited to the study of factors affecting hormonogenesis by the human placenta whether they be of maternal or of fetal origin

Detection of anti-pituitary autoantibodies by immunoblotting

A new approach to the detection of anti-pituitary autoantibodies by immunoblotting is presented. This method distinguishes pituitary membrane fraction from cytosolic fraction autoantigens and characterizes them by their molecular weight. A 45 kDa pituitary specific membrane protein was identified as an autoantigen in one of 19 patients with idiopathic growth hormone deficiency and the empty sella syndrome. A 43 kDa membrane protein in pituitary and brain was identified as an autoantigen in one other patient with idiopathic growth hormone deficiency and in one of 14 patients with secondary growth hormone deficiency. These autoantibodies were not seen in any of 27 control subjects. Anti-pituitary autoantibodies can be demonstrated by immunoblotting at titres of up to 1/1000. We conclude that immunoblotting is a useful method for the detection of anti-pituitary autoantibodies.

Human embryos produce transforming growth factors α activity and insulin-like growth factors II

Objective To assess whether growth factors are produced by early human embryos in culture. Design We studied various growth factors in the culture media of human embryos (n = 6) cultured from days 3 to 8 after fertilization. Main Outcome Measures Four growth factors were measured: Insulin growth factors I and II (IGF-I and IGF-II), epidermal growth factor (EGF) and transforming growth factor α (TGF α ) activity. Results Nonconditioned INRA Menezo B 2 (Biomerieux, S.A., Paris, France) culture medium contained significant levels of TGF α activity (5.2 ng/mL) and low levels of IGF-I (1.02 ng/mL) and IGF-II (2.8 ng/mL), whereas EGF was below detection of our assay. With human embryo, the culture media contained lower TGF α activity on days 3 and 4 after fertilization (2.5 ng/mL and 2.8 ng/mL, P α activity and IGF-II was detected (TGF α : activity: day 5: 3.7 ng/mL; day 6: 4.4 ng/mL; day 7: 6.4 ng/mL; day 8: 8.4 ng/mL) (IGF-II: day 5: 3.4 ng/mL; day 6: 3.1 ng/mL; day 7: 4.1 ng/mL; day 8: 4.2 ng/mL). Epidermal growth factor was undetectable, and IGF-I did not vary significantly. Conclusion Transforming growth factor α activity and IGF-II are produced by human embryos in culture at a time when they could play a role in morula to blastocyst transformation.