Radioimmunoassay For Prolactin Research Articles

Hyperprolactinemia Associated With Empty Sella Syndrome

SINCE the radioimmunoassay of human prolactin became available, hyperprolactinemia has been demonstrated in a number of pathological states.¹An elevated basal prolactin concentration of more than 200 ng/ml is generally regarded as strong evidence in favor of an adenomatous pituitary lesion.²Our patient had substantial hyperprolactinemia associated with the empty sella syndrome. <h3>Report of a Case</h3> A 37-year-old woman was referred to The Johns Hopkins Hospital in December 1973 because of persistent generalized headaches of five years' duration. Three years prior to admission, she underwent extensive examinations in another hospital. Skull roentgenograms had shown enlargement of the sella turcica, with erosion of the dorsum sellae (Fig 1). A right brachial arteriogram showed no abnormalities. Pneumoencephalography was attempted twice, but no air could be driven into the ventricular system (Fig 2). The patient was obese, normotensive, and without visual field defects. Galactorrhea could not be demonstrated. Serum thyroxine level

Seasonal variation in prolactin and TSH releasing activity in the hypothalamus of Rana temporaria

Radioimmunoassay of plasma prolactin (PRL) and thyroid stimulating hormone (TSH) has been performed after injection into rats of hypothalamic extracts obtained from R. temporaria. An increase in plasma prolactin followed the injection of an extract of the pars ventralis tuberis cinerei collected from frogs during April and May. A decrease was seen after the injection of hypothalami obtained during November and December. A decrease in plasma TSH was found with tuberal extracts collected during January, February, March and April, whereas no change was observed with extracts of tuberal tissue collected in other months. Comparisons of the TSH/PRL ratios after injections of the various extracts revealed ratios in favour of TSH release during fall caused by a decrease in prolactin; while prolactin was favoured in spring by both an increase in prolactin release and a decrease in TSH release. In a separate series of experiments, the releasing activities for prolactin also could be detected in extracts made from the preoptic area, but not from the median eminence. In these experiments also an increase in prolactin was seen following the injection of extracts collected during April and May, but not during June. It is concluded that the hypothalamus of R. temporaria contains factors which are able to influence both TSH and PRL secretion in the rat, and that these activities are not restricted to the pars ventralis tuberis cinerei. During the period of reproduction a PRF activity may facilitate life in water. Outside this period, PRF activity may disappear and be replaced by PIF activity. TSH secretion does not seem to be stimulated by a releasing factor, but an inhibitory activity is found during the period when PRF occurs.

Homologous radioimmunoassay for human prolactin

Radioimmunoassays for human prolactin in various biological fluids have been available since 1971. (1–7) Most of these assays have used heterologous systems with prolactin preparations from various sources, i.e. sheep, pig and primate with their corresponding antiserum. (1–5) Friesen et al. (6) find Sinha et al. (7) are the only authors who have described homologous assays using human prolactin as the reference standard and tracer as well and an anti-human prolactin antiserum. In the present study we have established a homologous radioimmunoassay using human pituitary prolactin. The separation method is based on the double antibody solid phase system. (8) Cross reactivity with human growth hormone (HGH), placental lactogen (HPL), the pituitary protein hormones and prolactins of various species were studied as were values found in normal subjects in basal conditions and after a TRH injection. The results of measuring basal levels and performing dynamic tests under various physiological and pathological circumstances will be reported subsequently.

Isolation and characterization of prolactin from the grey mullet, Mugil cephalus

1. 1. A prolactin-like hormone has been isolated from the pituitary of the grey mullet, Mugil cephalus (Linnaeus), by ultrafiltration and analytical chromatography on G-75 Sephadex. 2. 2. The hormone possessed biological activity in the Gillichthys xanthophore dispersion assay (Sage & Bern, 1971). 3. 3. A sensitive radioimmunoassay for mullet prolactin was developed and validated by comparison with biological activity data. Some immunological similarity between the ovine prolactin molecule and the mullet prolactin molecule was demonstrated by this radioimmunoassay. 4. 4. A molecular weight of 22,400 was indicated by the elution behavior of mullet prolactin from G-75 Sephadex.

Effects of intravenous infusion of catecholamines on rat plasma luteinizing hormone and prolactin concentrations.

Catecholamines were infused through an atrial cannula in unanesthetized rats on the afternoon of proestrus and blood was withdrawn through a second cannula for radioimmunoassay of LH and prolactin. Infusion of epinephrine, but not of norepinephrine or dopamine, blocked spontaneous pituitary LH release and ovulation. Ultimately, this effect appears to be exerted on the brain and not on the pituitary or through changes in pituitary blood flow. Pituitary LH release in response to exogenous LHRH, when administered in an amount that simulated the proestrous LH surge in phenobarital-treated rats, was unaltered by epinephrine infusion. In addition, epinephrine infusion did not alter the timing of the rise in plasma prolactin. Infusion of dopamine blocked the spontaneous rise in plasma prolactin and depressed basal prolactin levels. After the end of infusion, plasma prolactin rose rapidly. Infusion of norepinephrine or epinephrine partially suppressed the prolactin rise but only after 2 h of infusion. 1) point out the possibility that chronic release of epinephrine from the adrenal medulla may be involved in phenomena in which "stress" inhibits reproductive function; and 2) are consistent with the view that dopamine, but not norepinephrine, may be PIF.

The possible role of histamine in the control of prolactin and gonadotropin release.

To evaluate a possible role of histamine in the CNS control of prolactin and gonadotropin release, adult ovariectomized rats, with stainless steel cannulae implanted in the 3rd ventricle, were given s.c. injections of 10 mug of estradiol benzoate. 48 h later, 2 mul of 0.9% NaCl alone or of saline containing 1, 5, 25 or 125 mug of histamine dihydrochloride was microinjected into the ventricle. Immediately before and then 15, 30 and 60 min after, blood samples were withdrawn from etherized rats for radioimmunoassay (RIA) of serum prolactin, LH and FSH. In the histamine-injected rats, an increase in prolactin titers was observed and was highly significant in groups receiving the higher doses. A small yet significant release of LH, but not of FSH, was also observed. When 25 mug of histamine was injected directly into the pituitary or into the jugular vein, no elevations were observed, indicating a site of action in the brain. Restraint stress elevated serum prolactin and lowered serum LH and FSH in ovariectomized rats. These responses were blocked by the intrajugular injection of diphenhydramine (5 mg/kg). It is suggested that histamine may be involved in the hypothalamic control of prolactin release and possibly of gonadotropin release.

Radioimmunoassay and Radioreceptor Assays for Prolactin and Growth Hormone: A Critical Appraisal

The promise and the problems associated with using radioimmunoassays (RIA's) for mammalian prolactins (PRL's) and growth hormones (GH's) for the measurements of these hormones in the blood of foreign species (mammalian and nonmammalian) are considered. When crossreactivity is found with the plasma of a foreign species in heterologously applied RIA's for these hormones of mammalian origin, one can have little confidence about the nature of the crossreacting material that is being measured. Extensive analysis is necessary to establish that a particular RIA system measures the PRL or GH in a foreign species. The question of whether RIA's for PRL and GH give physiologically meaningful measurements of the blood levels of these hormones is considered. In the case of PRL, analysis of our own results and data in the literature raises serious questions about the physiological validity of existing RIA's for mammalian PRL's. Evaluation of the available information on RIA's for mammalian GH discloses that there is no basis for concluding that any of them give measurements of the circulating levels of the hormone that are physiologically meaningful. It is also apparent that the physiological significance of the radioreceptor assays for PRL and GH remains to be established. From analysis of discrepancies between bioassay and RIA estimates of PRL and GH levels in adenohypophysial tissue, incubation medium, and plasma or serum, and of studies on the metabolism of purified and secreted forms of both hormones, it is suggested that the major intraglandular forms of PRL and GH are in fact prohormones.

Radioimmunoassay of human prolactin (PRL).

A simple and rapid homologous radioimmunoassay of human prolactin (hPRL), by the use of 66% ethanol containing 6.6% ammonium acetate for the separation of free and bound hormones, has been established. PRL levels showed marked individual and daily variations as well as a circadian periodicity in both men and women. Levels of plasma PRL rose with puberty and decreased during post-menopause and in elderly men (ages 60-73 years). There was no marked change in plasma PRL levels at midcycle; however, in some subjects, luteal levels were slightly higher than follicular levels. Administration of oestradiol-17beta caused marked increase in plasma PRL levels during menstrual cycle. During pregnancy, the plasma PRL levels rose as high as 200 ng/ml and decreased within a week or two post-partum. During lactation, the levels were higher and increased with suckling. In a patient with habitual abortion, the plasma PRL level showed a sudden drop prior to abortion. Administration of LH-RH had no effect on the secretion of PRL in human subjects.

Prolactin levels and duration of postpartum anoestrus in lactating ewes.

In both lactating women and lactating ewes suckling is accompanied by large discharges of oxytocin and prolactin. The prolactin secretion decreases as lactation progresses being almost nonexistent by the time of return of cyclical ovarian activity. It has been suggested that prolactin has an antigonadal action. This study investigates ovarian and pituitary activities in lactating ewes after denervation of the mammary gland to inhibit the afferent arc of the suckling reflex. All nervous connections between the mammary gland and the body were severed in 5 Prealpes du Sud ewes during late pregnancy. The ewes were allowed to suckle their lambs after normal parturition as were 8 unoperated control ewes which delivered at the same time. Blood samples were drawn twice daily for radioimmunoassay of prolactin and luteinizing hormone (LH). Estrous behavior was checked after blood sampling using a vasectomized ram. All ewes were subjected to laparoscopy between days 12-15 25-31 and 40-50 postpartum for examination of ovarian status. The normal prepartum surge of prolactin secretion and basal levels during lactation were not affected by denervation but the suckling stimulus was not followed by the normal reflex discharge of prolactin. Control animals exhibited neither ovulation nor an LH peak before day 25 postpartum. Silent ovulations preceded by an LH surge occurred in 5 of 8 animals between days 25-50 postpartum; only 3 came into estrous during this period. In contrast 3 of 5 operated animals had ovulated without showing estrous before day 14 postpartum and all 5 animals had ovulated and shown estrous before day 50. The findings indicate that it is the surges of prolactin secretion induced by suckling which suppress the resumption of cyclical ovarian activity.

Hyperprolactinemia in sarcoidosis: incidence and utility in predicting hypothalamic involvement.

Patients with sarcoidosis have been reported frequently to have elevated concentrations of serum prolactin. On this basis, it was suggested that the hypothalamus might be a common site of involvement by sarcoidosis and that measurement of serum prolactin concentrations might serve as a sensitive indicator of hypothalamic disease. We measured serum prolactin concentrations in a group of 61 patients with sarcoidosis. Hyperprolactinemia was detected in only 2 of the entire group and was not observed in any of the 9 patients with central nervous system involvement. Because radioimmunoassayable prolactin concentrations are infrequently elevated in patients with disseminated sarcoidosis, even when pitutitary hypofunction is apparent, it is concluded that the measurement of serum prolactin is not a reliable method for screening these patients for pituitary or hypothalamic disease.

The clinical utility of a heterologous radioimmunoassay for human prolactin

A specific and sensitive double antibody radioimmunoassay for human prolactin has been developed utilizing anti-human prolactin and purified ovine prolactin for radioiodination. Employing this radioimmunoassay, as little as 1.5 ng of prolactin can be quantified in a ml of human serum. The prolactin in serum detected by the radioimmunoassay behaved in a similar manner to purified human 125 I-labeled prolactin on polyacrylamide gel electrophoresis. Exogenous prolactin could be completely recovered when varying amounts were added to a constant volume of serum. In normal control and primary hypothyroid subjects, intravenous administration of thyrotropin-releasing hormone elicited a peak prolactin response 30 min post-injection. Hyperthyroid and hypopituitary subjects did not manifest a significant increase in serum prolactin concentration after the injection. Subjects with functional galactorrhea had high resting prolactin levels and exhibited peak increase in serum prolactin concentration at 60 min following the administration of thyrotropin-releasing hormone.

Thyrotropin-releasing hormone blockade of the ergocryptine and apomorphine inhibition of prolactin release in vitro.

The interaction of thyrotropin-releasing hormone (TRH) and ergocryptine or apomorphine in affecting prolactin secretion was examined. Ergocryptine and apomorphine inhibited the release of 3H-prolactin labeled in an in vitro incubation of the rat anterior pituitary and led to a retention within the gland of 3H-prolactin. TRH (1.4 X 10(-8) M) partially blocked the inhibitory effect of 4 x 10(10) M ergocryptine on 3H-prolactin release and totally blocked the effect on radioimmunoassayable prolactin release. TRH also completely blocked the inhibitory effects of 5 x 10(-8) M apomorphine on both 3H-prolactin and radioimmunoassayable prolactin release. Since TRH can also completely block the inhibitory effects of dopamine upon prolactin secretion, these results are consistent with apomorphine and ergocryptine stimulating "dopaminergic" sites and also further support the role of TRH as a prolactin-releasing factor.

Hormone and glucose responses to serial cardiac puncture in rats (38465).

Serial, apparently basal radio-immunoassayable plasma insulin and growth-hormone values were obtained when fast-knock down ether anesthesia was used. Radioimmunoassayable prolactin was elevated at the second, third, and fourth bleedings but cause no apparent increase in circulating insulin levels. The elevation in plasma glucose may also have been a transient effect of sympathetic stimulation which simultaneous inhibited insulin release.

Studies on prolactin in human serum, urine and milk.

Prolactin activity was measured in serum, urine and milk using a specific human prolactin radioimmunoassay (RIA). Serum, urine and milk were parallel with the human prolactin standard in the RIA. There was no correlation between serum prolactin levels and urinary prolactin activity. Dialysis of urine samples resulted in complete loss of human prolactin activity while the addition of human prolactin to the urine resulted in the recovery of over 50% of the hormone after dialysis. Thus it was concluded that prolactin is not present in urine. In additional experiments it was observed that the RIA prolactin activity in urine was significantly correlated with the osmolality of the urine and that Na+ and K+ were contributory elements. On the other hand, prolactin was found in human milk and correlated well with the expected serum levels of this hormone. This latter finding is interesting because prolactin receptors have been shown to exist on the serosal side of the mammary epithelial cells. The presence of prolactin in milk suggests the possibility of other sites of action for this hormone in addition to the cell membrane.

PROLACTIN RELEASE IN THE SHEEP

The recent development of radioimmunoassay for prolactin has extended the depth and extent of studies of factors affecting prolactin levels in mammals and has permitted long-term studies to be made of changes in blood prolactin levels in individual animals. Although ovine prolactin has been widely studied in relation to its structure (Li, 1972) and biological activity (see Wolstenholme & Knight, 1972) and has been extensively used as a reference material, the

Modulation of prolactin release by altered levels of thyroid hormones

In an attempt to delineate the effect of thyroid hormones on prolactin release, serum concentrations of prolactin in response to synthetic thyrotropin-releasing hormone (TRH) administration in subjects with normal and altered levels of thyroid hormones were determined by a heterologous radioimmunoassay for human prolactin. In 16 male and 19 female control subjects, an intravenous administration of TRH elicited a definite elevation in prolactin levels having a peak at 15 min after injection. This response of prolactin was markedly attenuated in all of the ten untreated hyperthyroid patients but was restored in eight subjects after the treatment. In contrast, TRH-mediated prolactin release was enhanced in most cases of primary hypothyroidism. Replacement of thyroid hormones in these patients resulted in a decrease in prolactin response to TRH. Treatment of six euthyroid subjects with triiodothyronine, on the other hand, unequivocally suppressed the release of prolactin. These results suggest that thyroid hormones per se are responsible for the blunted response of prolactin secretion to TRH administration.

Report of the National Pituitary Agency. Collaborative study of the radioimmunoassay of human prolactin.

A collaborative study of the reagents for the radioimmunoassay of human prolactin (hPRL) to be distributed by The National Institute of Arthritis, Metabolism and Digestive Diseases was carried out. Each group applied the radioimmunoassay techniques and conditions currently used in their laboratory. The Lewis and Friesen hPRL tracers behaved identically though the former produced a more sensitive assay. Both hormones were iodinated satisfactorily by the Chloramine T method. The Friesen antiserum was more sensitive than the SLV antiserum. Similar results were obtained for each of the plasma samples supplied in the homologous and heterologous systems. The results obtained confirmed the reports of the investigators who donated the reagents.

DNA synthesis and the secretion of prolactin and growth hormone by the pituitary gland of the male rat: effects of diethylstilboestrol and 2-bromo-alpha-ergocryptine methanesluphonate.

SUMMARY The effects of 2-bromo-α-ergocryptine methanesulphonate on the response of the pituitary gland of the male rat to diethylstilboestrol dipropionate were studied by radioimmunoassay of prolactin and growth hormone and measurements of DNA synthesis. Given before diethylstilboestrol, 2-bromo-α-ergocryptine prevented the oestrogen-induced rise in serum prolactin and partly inhibited pituitary DNA synthesis. Given on days 4–6 of a 6-day period after a single dose of diethylstilboestrol, 2-bromo-α-ergocryptine rapidly increased pituitary prolactin concentration, diminished serum prolactin, reduced pituitary DNA synthesis and did not affect the growth hormone response to oestrogen.

Ultrastructural immunocytochemical localization of prolactin in rat anterior pituitary by use of the unlabeled antibody enzyme method.

The National Institute of Arthritis, Metabolism and Digestive Diseases antirat prolactin antiserum, used widely for radioimmunoassay of rat prolactin, was used in the unlabeled antibody-peroxidase-antiperoxidase complex method for the ultrastructural immunocytochemical staining of rat prolactin cells. Sections were exposed to antiprolactin antiserum (1:400-1:100,000) or to antiovine luteinizing hormone antiserum (1:1,000) for 3 min or 4 or 48 hr. Specificity of prolactin staining was demonstrated by the absence of immunologic staining of other pituitary cells and by negative results with prolactin cells when normal serum or antiovine luteinizing hormone antiserum was used. Intensity of prolactin staining was directly related to the concentration and duration of exposure to antiprolactin antiserum. Prolactin secretion granules, located peripherally or within Golgi regions, were uniformly stained. Immunologic staining for prolactin was not observed within cisternae of the endoplasmic reticulum. These results show that the National Institute of Arthritis, Metabolism and Digestive Diseases antirat prolactin antiserum is specific for prolactin by ultrastructural immunocytochemical criteria and that antiserum dilutions appropriate for radioimmunoassay can be used in immunologic localization studies.

Studies on the mechanism of the dopamine-mediated inhibition of prolactin secretion.

A study of the in vitro effect of dopamine on the release of newly synthesized prolactin is reported. Pituitary glands of female rats were incubated with 4,5-3H-leucine and the radioactive prolactin present in the pituitary gland and that released into the incubation medium were measured. Incubation with 5 × l 10–7M dopamine caused an 85% decrease in prolactin release. Prior injection of the rats with perphenazine or haloperidol rendered the pituitary gland refractory to the in vitro inhibitory effect of dopamine. Although in vitro perphenazine and haloperidol had little or no effect on release of prolactin, 5 × 10–9M of these drugs directly blocked the in vitro action of dopamine on prolactin release. Phentolamine, an α-blocking agent, was partially able to block the inhibitory effect, of dopamine. Propranolol, however, was not effective. Apomorphine, an agent known to mimic the effects of dopamine, caused a significant decrease in the amount of radioactive and radioimmunoassayable prolactin released int...