Discrimination between beta-endorphin and beta-lipotrophin in human plasma using two-site immunoradiometric assays.

OBJECTIVE: We wished to discriminate between the opioid peptide beta-endorphin (beta-EP) and its non-opioid precursor beta-lipotrophin (beta-LPH) in normal subjects and patients with ACTH-related disorders. DESIGN: We produced monoclonal antibodies to beta-EP and beta-LPH for the development of two-site immunoradiometric assays (IRMAs) which specifically quantitate beta-EP and beta-LPH. PATIENTS: Samples were obtained from patients with a range of ACTH-related disorders and compared with 18 normal subjects. Peptide levels were also compared in six patients with Cushing's syndrome undergoing bilateral inferior petrosal sinus sampling with corticotrophin releasing hormone administration. MEASUREMENTS: In the beta-EP IRMA, antibody 6B2, specific for beta-EP 18-27, is radiolabelled and antibody 2E10, recognizing beta-EP 1-5, is coupled to Sephacryl S-300 as solid phase. The IRMA is specific for beta-EP (beta-LPH cross-reacts < 0.02%), has a detection limit of 1.4 +/- 0.7 pmol/l (n = 7) and has a within-assay coefficient of variation of < 10% between 4.9 and 1200 pmol/l. In the beta-LPH IRMA, antibody 6B2, which recognizes an epitope common to beta-LPH and beta-EP, is radiolabelled and paired with solid-phase antibody 5C11 which recognizes beta-LPH 39-56. The binding site of this antibody ensures that beta-EP cannot be measured in the beta-LPH assay. The detection limit is 0.8 +/- 0.1 pmol/l (n = 9) and the within-assay coefficient of variation is < 10% at concentrations 1.7-870 pmol/l. RESULTS: In normal subjects, beta-EP and beta-LPH levels were < 1.4-1.7 pmol/l (< 5-6 ng/l) and 2.5-6.7 pmol/l (29-77 ng/l) at 0930 h and < 1.4-1.7 pmol/l (< 5-6 ng/l) and 1.9-4.5 pmol/l (22-49 ng/l) at 1600 h, respectively. In patients with ACTH-related pathologies concentrations of beta-EP and beta-LPH paralleled those of ACTH. The ratio of beta-LPH:beta-EP in plasma varied between 3.2:1 and 38:1 in these patients demonstrating that beta-LPH is the major circulating peptide derived from the C-terminal of pro-opiomelanocortin in man. However, in two patients undergoing bilateral inferior petrosal sampling with corticotrophin releasing hormone for diagnosis of Cushing's disease beta-EP concentrations increased rapidly during the first 5 minutes of the test, resulting in a sharp decrease in the beta-LPH: beta-EP ratio. These results suggest that beta-EP is preferentially released in response to acute corticotrophin releasing hormone stimulation. CONCLUSIONS: It is concluded that two-site IRMAs for beta-EP and beta-LPH provide an easy approach to study the dynamic changes in processing of beta-LPH to beta-EP.

Altered ratios of beta-endorphin: beta-lipotropin released from anterior lobe corticotropes with increased secretory drive. I. Effects of diminished glucocorticoid secretion.

Previous studies have demonstrated that acute stress or ovine corticotropin-releasing hormone (oCRH) in vivo, or oCRH in vitro, stimulates release of beta-endorphin over beta-lipotropin from anterior pituitary corticotropes. This occurs despite the predominance of beta-lipotropin in corticotrope peptide stores. In vitro studies with primary anterior pituitary cultures suggested that chronic exposure to oCRH results in a shift towards more beta-lipotropin secretion into the media than with short-term exposure. The current studies explored whether increased secretory drive in vivo results in a similar shift towards more beta-lipotropin. We used removal of glucocorticoids by adrenalectomy or metyrapone blockade of corticosterone synthesis, to stimulate endogenous secretion of CRH and vasopressin. Both treatments resulted in shifts of the ratio of beta-endorphin: beta-lipotropin in plasma of experimental animals in comparison to the sham-treated control rats. In vitro testing with oCRH of anterior lobe cultures from adrenalectomized or metyrapone-treated rats demonstrated similar effects of these treatments on the ratio of beta-endorphin:beta-lipotropin. These changes occurred despite similar ratios of beta-endorphin:beta-lipotropin in anterior pituitary peptide stores.

Altered ratios of beta-endorphin:beta-lipotropin released from anterior lobe corticotropes with increased secretory drive. II. Repeated stress.

A number of stimuli including acute footshock and electrically-induced seizures lead to release of beta-endorphin immunoreactivity from the anterior pituitary corticotropes. Gel filtration of this beta-endorphin immunoreactivity indicates that approximately 3-fold more beta-endorphin than beta-lipotropin is released into plasma following these acute stressors. A similar preponderance of beta-endorphin over beta-lipotropin is seen in the media of short-term anterior lobe cell suspensions stimulated with ovine corticotropin-releasing hormone. Previous studies indicated that footshock stress, when administered repeatedly, can increase the biosynthesis of anterior lobe proopiomelanocortin (POMC) as indicated by increased steady state adrenocorticotropin/beta-endorphin content as well as increased POMC mRNA levels and increased POMC biosynthesis and rate of processing as measured by pulse-labeling and pulse-chase studies. The goal of the present studies was to determine whether this increased biosynthetic drive results in an alteration in the end products secreted with repeated stress. Acute footshock in a rat which has received 14 days of chronic footshock releases proportionately more beta-lipotropin than is released in a naive rat. Chronic electrically-induced seizures, which also increase anterior lobe POMC derived peptide stores, lead to a similar shift in the ratio of beta-lipotropin:beta-endorphin released following stress. These data suggest that chronic drive and the subsequent changes in POMC peptide stores may lead to a decrease in the proportion of beta-endorphin size immunoreactivity in the releasable pool of the anterior lobe corticotrope, thus altering the hormonal signal from the anterior lobe corticotrope.

Beta-lipotropin-like immunoreactivity in intraglandular colloid from pituitary intermediate lobe cells.

The aim of this paper is the identification of beta-lipotropin (beta/LPH) as a peptide present in intraglandular colloid (the holocrine secretion of cells in the marginal half of the bovine pituitary intermediate lobe). beta/LPH, although not an opioid peptide itself, contains the peptide beta-endorphin. The methodology used allowed detection of beta/LPH when present in the samples in sufficient amounts.

Plasma pro-opiomelanocortin fragments and adrenal steroids following administration of metyrapone to normal and hirsute women.

The present study was undertaken to characterize further the adrenal abnormalities in the polycystic ovary syndrome (PCOS) and idiopathic hirsutism (IH). We have previously reported the close association of elevated estrone levels with amenorrhea in hyperandrogenemic patients. In addition we have suggested that high estrone levels in PCOS occur as a consequence of the provision of excess substrate, androstenedione, for conversion to estrone. In the present study plasma estrone levels rose following endogenous adrenal stimulation induced using metyrapone; the highest plasma estrone levels achieved were seen in patients with PCOS and occurred later than peak androstenedione levels. These findings are consistent with the hypothesis that elevated estrone levels occurring in PCOS may arise as a consequence of increased adrenal androgen secretion. In addition, we have examined ACTH and other pro-opiomelanocortin (POMC) fragments in an attempt to identify a factor responsible for the excessive adrenal androgen secretion occurring in hirsute patients. Plasma levels of ACTH, and of immunoreactive beta-endorphin and h-lipotropin were not elevated when androgen levels were raised prior to therapy, although these POMC fragments, and also the 16K fragment, rose in response to metyrapone treatment as did androgen levels. Following treatment with dexamethasone there was more profound suppression of the 16K, beta-endorphin and h-lipotropin responses to metyrapone stimulation, than of the ACTH response, as indicated by decreased POMC-fragment/ACTH ratios; this parallels the dissociation of cortisol from androgens in hirsute patients under similar conditions. However, we have not identified a POMC fragment which consistently parallels changes in androgen levels in patients with idiopathic hirsutism or PCOS.

Immunolocalization of beta-lipotropin in the inner ear of the guinea pig.

The occurrence of beta-lipotropin (beta-LTH) immunoreactive material was investigated in the inner ear of newborn and juvenile guinea pigs by means of Sternberger's PAP technique. Unlike met5-enkephalin and endorphin, beta-LTH could not be found in the organ of Corti but was identified in the spiral ganglion and the neuroepithelium of the crista ampullaris.

beta-Endorphin/beta-lipotropin immunoreactivity in endogenous depression. Effect of dexamethasone.

This study addresses the question of whether pituitary peptides (ie, beta-endorphin) show regulatory disruption in endogenous depression and, if so, does it co-occur in the same subjects who show cortisol dysregulation. Endogenously depressed patients and psychiatric controls from three centers were evaluated, when not taking medications, and studied for plasma cortisol and beta-endorphin levels. Plasma samples were taken at four time points over one hour, on the basal day, and 16 hours after 1 mg of dexamethasone. From 33% to 69% of the endogenous patients were abnormal in their postdexamethasone cortisol levels, and from 50% to 69% were abnormal on postdexamethasone beta-endorphin values (vs 0% and 8%, respectively, for controls). When endogenous subjects were evaluated for abnormality on both cortisol and beta-endorphin, after dexamethasone, it was found that the two measures of hypothalamic-pituitary-adrenal dysfunction did not necessarily co-vary. In fact when having either abnormal beta-endorphin or cortisol levels (or both) was used as a biological marker a larger number of the endogenous patients were detected than with either measure alone. Our conclusions are as follows: Plasma beta-endorphin shows a circadian rhythm similar to that seen with corticotropin (ACTH) and is suppressable by dexamethasone. In many endogenous patients plasma beta-endorphin levels escape from dexamethasone suppression. Many of these subjects are not cortisol escapers. When abnormality of either the beta-endorphin or cortisol is considered it is clear that both levels of the hypothalamic-pituitary-adrenal axis can be dysregulated in endogenous depression.

Cell types of the pars distalis of the hedgehog (Erinaceus europaeus L.) adenohypophysis: cytological, immunocytochemical and ultrastructural studies. 3. Opiocorticomelanotropic cells.

Staining and histochemical methods, immunofluorescence and electron microscopy were used to individualize the opiocorticomelanotropic cells in the adenohypophysis of the non-hibernating hedgehog. One cell type was differentiated; its characteristics at the electron-microscopic level were presented. Immunofluorescence has confirmed the functional significance of this cell type and the validity of the denomination 'opiocorticomelanotropic cells'.

Rapid and specific radioimmunoassays for beta-endorphin and beta-lipotropin in affinity-purified human plasma.

Using rapid and reusable affinity columns, we established a method to measure beta-endorphin (beta END) and beta-lipotropin (beta LPH) separately in human plasma. One column contained antibodies against the N-terminal portion of beta LPH, and the other contained antibodies against beta END. The first column separated beta LPH from beta END and concentrated beta LPH as well, and the second separated beta LPH from concentrated beta END. Mean plasma levels (at 0830-0930 h) of beta END and beta LPH in 10 normal subjects were 1.1 +/- 0.1 (+/- SE) and 4.1 +/- 0.4 fmol/ml, respectively; both were markedly elevated in patients with ACTH/LPH hypersecretory states. The molar ratios of plasma levels of beta END and beta LPH were fairly constant in normal subjects (0.28 +/- 0.01), unchanged in patients with Cushing's disease and Cushing's disease after adrenalectomy, lower in patients with Addison's disease (P less than 0.001), higher in patients with chronic bone pain (P less than 0.001), and variable in patients with the ectopic ACTH syndrome. These data indicate that beta END and beta LPH can be measured separately in plasma by simple and reproducible procedures.

Maternal and umbilical venous plasma immunoreactive beta-endorphin levels during labor with and without epidural analgesia.

Maternal and umbilical venous plasma immunoreactive beta-lipotropin/beta-endorphin levels were determined during labor in 23 healthy parturient women at term. Eleven of the mothers received a segmental epidural analgesic for relief of pain, whereas the other 12 mothers were nearly pain-free and needed no analgesia. Maternal immunoreactive beta-lipotropin/beta-endorphin levels were already significantly elevated at the beginning of labor in both groups in comparison with nonpregnant young women. Maximum levels of immunoreactive beta-lipotropin/beta-endorphin were reached at delivery, and these mean levels were significantly higher than the initial mean levels in the epidural group (p less than 0.05) and in the control group (p less than 0.001). There were statistically no significant differences between the groups at any time. The umbilical venous plasma immunoreactive beta-lipotropin/beta-endorphin levels did not differ from each other in the epidural and the control groups. These results suggest that the stress of labor causes an increase in the maternal secretion of immunoreactive beta-lipotropin/beta-endorphin which is not related to the degree of pain itself. Epidural analgesia has also no effect on umbilical venous plasma immunoreactive beta-lipotropin/beta-endorphin.

Elucidation of the antigenic determinant in lipotropin for two monoclonal antibodies.

Two monoclonal antibodies were found to give enhanced affinity for beta-lipotropin when mixed, as evidenced by competitive radioimmunoassay. Both monoclonals were found to react with a pentapeptide Ala-Glu-Leu-Glu-Tyr, which is a sequence of high local hydrophilicity within the N-terminal section of beta-lipotropin.

Radioimmunological measurement of beta-endorphin in equine plasma.

Radioimmunoassay procedures were developed and validated for the quantification of beta-endorphin (beta-EP)-like immunoreactivity in equine plasma. beta-EP could be quantitatively extracted from plasma with silicic acid powder and subsequently assayed, however, valid estimates of this hormone could also be obtained on unextracted plasma. Although beta-lipotropin (beta-LPH) cross-reacted in the assay, it was not necessary to correct for beta-LPH activity when assaying unextracted plasma because chromatographic analyses showed that 92% of the immunoreactivity in plasma extracts was similar in molecular weight to authentic beta-EP (1-31). In addition, electroacupuncture treatment did not alter the relative proportion of immunoreactivity among different molecular weight fractions.

The induction of antibodies to human luteinizing hormone by contaminated clinical pituitary hormone preparations.

Three children with hypopituitarism had elevated LH levels measured by RIA which were incompatible with their stage of sexual maturation. Each of the children had been administered parenteral pituitary hormone preparations: one patient, human (h) GH for 3 2/1 yr; one patient, bovine TSH twice to evaluate thyroid responsiveness; and two patients, posterior pituitary extract by nasal insufflation for 7 5/12 ad 4 9/12 yr to treat diabetes insipidus. Each of these children had developed antibodies of the immunoglobulin G class which bound [125I]hLH in vitro in a displaceable fashion. In two of the patients, the antibody reacting with hLH was found after therapy with pituitary hormones of bovine or porcine origin and before treatment with hGH, while on child had received only hGH therapy. These antibodies interfered in the assay for hLH and were responsible for the spurious elevations of serum immunoreactive hLH. None of these children had undergone spontaneous puberty, including at least one who may not have been gonadotropin deficient. To reduce the risk of generating high potency neutralizing antibodies, only highly purified, monomeric pituitary hormone preparations or pure synthetic hormone preparations should be used for diagnosis and chronic replacement therapy.

Detection and affinity purification of beta-endorphin precursors using a monoclonal antibody.

A monoclonal antibody to porcine beta-lipotropin has been produced which binds to the N-terminal (gamma-lipotropin) portion of the molecule. The antibody can be used to detect beta-lipotropin as well as other beta-endorphin precursors (predominantly a Mr 38 000 polypeptide) using radiobinding assay or the immunoblotting technique. Purification of the peptides can be readily achieved by affinity chromatography using the monoclonal antibody covalently bound to Sepharose 4B. As the antibody recognises the N-terminal part of beta-lipotropin, it can be used to detect and purify beta-lipotropin and other beta-endorphin precursors in the presence of beta-endorphin.

Human gamma-lipotropin radioimmunoassay: identification of immunoreactive gamma-lipotropin in human plasma and tissue.

Disagreement exists concerning the relative contributions to total plasma immunoreactive human lipotropin (hLPH) made by h beta LPH and its amino-terminal fragment, h gamma LPH [h beta LPH-(1-58)]. Using an antiserum (R1547) which requires the free COOH-terminal Asp58 residue of h gamma LPH for full affinity and reacts only 1% as well with h beta LPH and antisera (R3 and G106) that react with both LPHs, we examined gel chromatography eluate fractions of plasma extracts from one normal subject under basal conditions and another after metyrapone administration, of plasma or plasma extracts from patients with ACTH/LPH hypersecretion of various causes, of an extract two normal pituitary glands, and an extract of an ectopic ACTH/LPH-secreting tumor. Immunoreactive h gamma LPH was always a major, frequently the predominant, and sometimes the only immunoreactive LPH observed. We also observed in plasma or tissue of two patients with ectopic ACTH/LPH syndrome a peptide whose immunoreactivity and apparent molecular size were consistent with those of octadecapeptide human beta MSH, a molecule that is not thought to exist in normal man. These studies demonstrate that h gamma LPH is a major LPH component in plasma and tissues and indicate that the h gamma LPH RIA provides a reliable estimate of the h gamma LPH concentration in plasma without prior chromatography.

Vasopressin stimulates release of beta-lipotropin and beta-endorphin in conscious rats as measured by radioimmunoassay of unextracted plasma.

Using a newly developed radioimmunoassay to determine the beta-endorphin-like immunoreactivity (beta-EI) in unextracted plasma, the effect of vasopressin injections on plasma beta-EI was investigated in conscious rats. Arginine vasopressin caused a dose-dependent increase of plasma beta-EI from 34.5 to 7.8 fmol ml--1 (n = 6) in vehicle-treated animals to 205.0 +/- 36.1 fmol ml--1 (n = 7) after injection of the highest vasopressin dose employed (486 ng/100 g b.w.). In view of the appreciable cross-reactivity of beta-lipotropin (beta-LPH) in the radioimmunoassay used, plasma was extracted and subjected to gel chromatography on a Sephadex G-50 column. On average, about 70% of the beta-EI co-eluted with human beta-LPH and about 30% with human beta-endorphin in plasma extracts obtained from both control and vasopressin-treated rats. No peripheral conversion of human beta-LPH occurred under the experimental conditions, since after i.v. bolus injection of human beta-LPH 97% of the beta-EI comigrated with human beta-LPH during gel filtration. A similar blood pressure increase to that induced by the vasopressin injections, when elicited by noradrenaline or angiotensin II i.v., was not followed by an elevation of plasma beta-EI. These data indicate that vasopressin stimulates beta-lipotropin and beta-endorphin release into the systemic circulation in vivo.