Human Beta-lipotropin Research Articles

Effects of bile salts on plasma concentration of beta-endorphin-like immunoreactivity in men.

The effects of bile salts on the release of beta-endorphin-like immunoreactivity (beta-END-LI) were investigated in men using a specific radioimmunoassay developed by the authors. Plasma beta-END-LI was determined after extraction by the acid-acetone method (recovery: 73 +/- 5%). Oral administration of 400 mg of sodium taurocholate caused a rise in plasma beta-END-LI from 9.9 +/- 0.5 pmol/liter to 21.3 +/- 1.2 pmol/liter after 30 min and 18.1 +/- 0.5 pmol/liter after 60 min, with return to the initial value after 90 min. Oral administration of chenodeoxycholic acid (CDCA) also increased plasma beta-END-LI from a basal level of 8.4 +/- 0.7 pmol/liter to 18.7 +/- 0.8 pmol/liter after 30 min. Oral administration of ursodeoxycholic acid (UDCA) increased plasma beta-END-LI from 7.3 +/- 0.3 pmol/liter to 30.6 +/- 0.2 pmol/liter after 30 min. In gel chromatography, the beta-END-LI released after UDCA administration separated into two components, which eluted in the same positions as human beta-lipotropin and human beta-endorphin, respectively. These results suggested that bile salts may participate the release of beta-END-LI.

The effects of corticotropin, opioid peptides and crude pituitary extract on the production of dehydroepiandrosterone and corticosterone by mature rat adrenal cells in tissue culture

In order to study the steroidogenic response to pituitary factors, a technique of monolayer tissue culture of mature female rat adrenal cells was used. During the first 24 h, rat adrenal cells produced dehydroepiandrosterone (DHEA) and small amount of corticosterone but in the absence of corticotropin (ACTH), the release of these two steriods were reduced to very low levels. The addition of synthetic α-ACTH-(1–24)[0.01–100ng/ml] elicited a marked increase in the production of both steriods. This stimulating effect was not observed when synthetic methionine and leucine-enkephalins (1–100 ng/ml), human beta-endorphin (1–100 ng/ml) or human betalipotropin (1 ng/ml), were added to the culture medium. When these peptides were added concomitantly with α-ACTH (1–24) at half of the maximum response dose (1 ng/ml), no synergistic effect upon DHEA and corticosterone production was shown. The addition of crude extract from rat pituitary gland (1–100 ng/ml) with or without α-ACTH-(1–24) definitely showed both a stimulatory and synergistic effect upon the production of these two steroids. Furthermore, the ratio between DHEA production and corticosterone production was significantly higher when crude extract of the pituitary gland was given alone or concomitantly with α-ACTH(1–24) than when α-ACTH(1–24) was given alone. These data suggest the existence of a still undefined pituitary adrenal adrogen stimulating which may preferentially stimulate DHEA production over corticosterone production.

Effects of beta-lipotropin, beta-endorphin, gamma 2-melanotropin and corticotropin on steroid production by isolated human adrenocortical cells.

Normal human adrenocortical cells (from multi-organ donors) were incubated with corticotropin (tetracosactide), highly purified human beta-lipotropin, synthetic human beta-lipotropin, gamma 2-melanocyte stimulating hormone and beta-endorphin. Corticotropin stimulated cortisol, aldosterone and 18-hydroxycorticosterone production starting at 10(-12)-10(-11) mol/l in normal adrenocortical cells. Purified human beta-lipotropin also stimulated steroidogenesis but 100-1000-fold higher concentrations of the peptide were needed. In contrast, synthetic human beta-lipotropin was without any effect on steroid production up to concentrations of 10(-7) mol/l. Synthetic beta-lipotropin (5 x 10(-10) mol/l) did not significantly change the dose-response curve for corticotropin (10(-13) mol/l-10(-9) mol/l) versus the three steroids measured. gamma 2-Melanotropin and beta-endorphin (10(-6) mol/l) stimulated the secretion of cortisol, but not of aldosterone. Since synthetic human beta-lipotropin has no effect on human adrenocortical cells, the purified beta-lipotropin must be contaminated with traces of corticotropin. Since pathologically elevated levels of proopiomelanocortin-derived peptides will rarely exceed plasma concentrations of 10(-10) mol/l, our findings in vitro do not support a physiological or pathophysiological role of the peptides examined in the regulation of adrenal steroid secretion.

Kinetic studies on the processing of human beta-lipotropin by bovine pituitary intermediate lobe pro-opiomelanocortin-converting enzyme.

The kinetics of the previously reported paired basic residue-specific pro-opiomelanocortin-converting enzyme from bovine pituitary intermediate lobe secretory vesicles (Loh, Y. P., Parish, D.C., and Tuteja, R. (1985) J. Biol. Chem. 260, 7194-7205) were studied using human 125I-beta-lipotropin as substrate. The enzyme, at a concentration of 20 ng/100 microliters cleaved human beta-lipotropin to yield gamma-lipotropin, a beta-melanotropin linked to beta-endorphin intermediate and beta-endorphin, whereas at an enzyme concentration of 40 ng/100 microliters, the substrate was completely cleaved to yield beta-endorphin and beta-melanotropin. These products were identified by their immunological properties and size on sodium dodecyl sulfate-polyacrylamide gels. The 125I-beta-endorphin product was further shown by high pressure liquid chromatography to contain two forms; the major form co-eluted with 125I-Arg0-beta h-endorphin and the minor form with 125I-beta h-endorphin. No COOH-terminal shortened forms of beta-endorphin were detected. The products formed indicate cleavages at two of the three pairs of basic residues of human beta-lipotropin, at Lys37-Lys38 and Lys57-Arg58, but not at Lys86-Lys87. The cleavage at Lys57-Arg58 occurred primarily in between these basic residues. The Km values for the cleavage of the Lys37-Lys38 and Lys57-Arg58 pairs were 1.9 and 2.5 microM, respectively. The Vmax values for the cleavage of the Lys37-Lys38 and Lys58-Arg58 pairs were 4.8 nmol/micrograms of enzyme/h and 9.1 nmol/micrograms of enzyme/h, respectively.

In vivo and in vitro effects of histamine on the release of beta-endorphin-like immunoreactivity

The effect of histamine on the release of beta-endorphin-like immunoreactivity (beta-END LI) in rats was studied in vivo and in vitro experiments. Intravenous injection of 100 micrograms/100g BW of histamine resulted in a significant increase in the plasma beta-END-LI level 5, 15 minutes after the injection. Histamine at concentrations of 10(-12) to 10(-9)M also caused dose-dependent stimulation of release of beta-END-LI from the dispersed cells of the anterior pituitary of rats. On gel-chromatography, the beta-END-LI released by incubating the cells with 10(-9)M histamine consisted of two components, which eluted in the same positions as human beta-lipotropin and human endorphin, respectively. Addition of 2mM CoCl2 to the incubation medium inhibited histamine-induced beta-END LI release from the cells. Histamine H1 receptor antagonist (10(-6)M) inhibited histamine-induced beta-END-LI release from the cells. Histamine H2 receptor antagonist (10(-6)M), however, did not inhibit histamine-induced beta-END-LI release. These results indicate that histamine acts directly on the anterior pituitary cells to stimulate beta-END-LI release and that calcium ion is involved in the mechanism of this effect.

Evidence that beta-endorphin binds to specific receptors in rat peripheral tissues and stimulates the adenylate cyclase-adenosine 3',5'-monophosphate system.

With the use of [125I]acetyl human beta-endorphin (Ac-hBE), specific binding sites for beta-endorphin (BE) were identified in the liver, kidney, adrenal, spleen, and testis of adult male rats, whereas specific BE-binding sites were not present in the ventral prostate or pancreas. In those tissues containing specific BE-binding sites, microsomal membranes (15,000-100,000 X g pellet) exhibited higher BE-binding capacity than the crude homogenate (125-100,000 X g pellet). The binding of BE was saturable, and maximal, specific binding was achieved with a 60-min incubation at 22 C. Furthermore, optimal BE binding was dependent on the presence of magnesium chloride. Scatchard analysis of BE binding to hepatic membranes revealed the existence of two classes of binding sites. One class had an apparent Ka of 0.019 X 10(9) M-1 and a lower number of binding sites (9.1 pmol BE/mg protein), whereas the other class had a lower affinity (apparent Ka of 0.0006 X 10(9) M-1) and a higher number of binding sites (159 pmol/mg protein). Specific BE binding to hepatic membranes was inhibited (80-100%) by rat AcBE-(1-27) and -(1-31), nonacetylated rat BE-(1-31), and human beta-lipotropin. At substantially higher peptide concentrations (greater than 10(-5) M), gamma-endorphin, met-enkephalin, or leu-enkephalin inhibited BE binding by 20-40%. In addition, opiate receptor-binding drugs, such as morphine and naloxone, at 10(-5) M did not alter BE binding to hepatic membranes. Incubation of hepatic membranes with BE induced a dose-related increase in membrane adenylate cyclase activity, and 0.5 X 10(-10) M BE resulted in a maximal enhancement of adenylate cyclase activity to 148% above control values. Water-deprived or salt-loaded male rats with chronically lowered immunoreactive plasma BE exhibited substantially increased BE binding to adrenal and kidney tissue. Specific binding sites for BE occur in a variety of peripheral tissues, and alterations of circulating BE result in changes in the capacity of certain peripheral tissues to bind BE. Finally, occupancy of specific BE-binding sites in peripheral tissue stimulates the adenylate cyclase-cAMP system, which suggests that the peripheral actions of circulating BE may be mediated via this system.

In vivo and in vitro effects of bradykinin on the release of beta-endorphin-like immunoreactivity.

The effect of bradykinin (BK) on the release of beta-endorphin-like immunoreactivity (beta-EpLI) in rats was studied in vivo and in vitro. Intraperitoneal injection of BK at 5 micrograms/100 g body weight resulted in significant increase in the plasma beta-EpLI level after 15 min. BK at concentrations of 10(-12)-10(-7) M also caused dose-dependent stimulation of beta-EpLI release from dispersed cells of rat anterior pituitary. On gel chromatography, the beta-EpLI released by incubation of the cells with 10(-7) M BK separated into two components, eluted in the same positions as human beta-lipotropin and human beta-endorphin, respectively. BK did not stimulate beta-EpLI release in Ca++-free medium. Addition of 10(-3) M verapamil or 10(-6) M dexamethasone to the incubation medium inhibited BK-induced beta-EpLI release from the cells. Quabain (10(-5) M) also stimulated beta-EpLI release, but its effect was not additive with that of BK. These results indicate that BK stimulates beta-EpLI release and that calcium ion is involved in the mechanism of this effect.

In vivo and in vitro effects of bradykinin on the release of beta-endorphin-like immunoreactivity

The effect of bradykinin (BK) on the release of beta-endorphin-like immunoreactivity (beta-END-LI) in rats was studied in in vivo and in vitro. Intraperitoneal injection of BK at 5 micrograms/100 g body weight resulted in a significant increase in the plasma beta-END-LI level after 15 min. BK at concentrations of 10(-12)-10(-7) M also caused dose-dependent stimulation of beta-END-LI release from the dispersed cells of the anterior pituitary of rats. On gel chromatography, the beta-END-LI released by incubation of the cells with 10(-7) M BK separated into two components; one eluted in the same positions as human beta-lipotropin and the other as human beta-endorphin. BK did not stimulate beta-END-LI release in Ca++-free medium. Addition of 10(-3) M verapamil, 10(-6) M dexamethasone or 10(-7) M somatostatin to the incubation medium inhibited BK-induced beta-END-LI release from the cells. Ouabain (10(-5) M) also stimulated beta-END-LI release, but its effect was not additive with that of BK. These results indicate that BK stimulates beta-END-LI release and that calcium ion is involved in the mechanism of this effect.

In vivo and in vitro effects of angiotensin II on the release of beta-endorphin-like immunoreactivity.

The effect of angiotensin II (A II) on the release of beta-endorphin-like immunoreactivity (beta-END-LI) in rats was studied in vivo and in vitro. Intravenous injection of 1 microgram/100 g body weight of A II resulted in significant increase in the plasma beta-END-LI level after 10 and 20 min. Intraventricular injection of 1 ng/100 g body weight of A II also resulted in significant increase in the plasma beta-END-LI level after 10 min. A II at concentrations of 10(-12) M-10(-10) caused dose-dependent stimulation of beta-END-LI release from dispersed cells of rat anterior pituitary. On gel chromatography, the beta-END-LI released by incubation of the cells with 10(-10) M A II separated into two components which eluted in the same positions as human beta-lipotropin and human beta-endorphin, respectively. The ratio of beta-LPH to beta-END in these fractions was 5:1 on a molar basis. A II did not stimulate beta-END-LI release in Ca++-free-medium. [Sar1, Ala8]-A II at concentrations of 10(-9) M - 10(-7) M did not stimulate beta-END-LI release from the cells. Addition of [Sar1, Ala8]-A II to the incubation medium inhibited A II-induced beta-END-LI release from the cells. These results indicate that A II acts, at least in part, directly on anterior pituitary cells to stimulate beta-END-LI release and that calcium ion is involved in the mechanism of this effect.

Binding characteristics of a monoclonal beta-endorphin antibody recognizing the N-terminus of opioid peptides.

The present paper describes the isolation and characterization of a clone of hybrid myelomas (3-E7) secreting a mouse monoclonal antibody to beta-endorphin. An examination of its specificity against a series of human beta-lipotropin fragments and other opioid peptides revealed that the N-terminus portion of beta-endorphin is the determinant. Complete or almost complete cross-reactivity was obtained to methionine- and leucine-enkephalin, beta-lipotropin 60-65, and BAM 22; partial cross-reactivity was seen to dynorphin1-13 and alpha-neo-endorphin, whereas beta-lipotropin, alpha-N-acetyl-beta-endorphin, Des-Tyr1-beta-endorphin, in addition to a series of synthetic enkephalin derivatives, completely lacked cross-reactivity. The use of the monoclonal antibody in radioimmunoassay (RIA) for beta-endorphin resulted in a lower sensitivity related to respective polyclonal antibodies. An increase of 100% in tracer binding could, however, be obtained by use of beta-endorphin iodinated with its N-terminal tyrosine protected by coupling to an antibody. A solid-phase RIA was developed involving the internally 3H-labeled monoclonal antibody, which resulted in a 10-fold increase in sensitivity as compared with the homogenous RIA. These data indicate that for the binding to this antibody a tyrosine residue in position 61 is essential, and it thus recognizes a site that is of functional significance for many naturally occurring opioid peptides.

Total synthesis of human beta-lipotropin.

The total synthesis of human beta-lipotropin has been accomplished by the new segment-coupling method in aqueous solution. The peptides Ac-Arg-beta-lipotropin-(61-89) (I) and [GlyS60]-beta-lipotropin-(1-60) (II) were synthesized by the solid-phase method. Reaction of peptide I with citraconic anhydride followed by brief digestion with trypsin to remove the acetylarginyl group, gave Ia. Reaction of peptide II with citraconic anhydride gave the citraconyl peptide IIa. Ia and IIa were coupled together in 50% dimethylformamide by reaction with silver nitrate/N-hydroxysuccinimide. After removal of the citraconyl groups in 25% acetic acid, a 10% yield of synthetic beta-lipotropin could be isolated. The synthetic product was shown to be identical to native human beta-lipotropin by paper electrophoresis, isoelectric focusing, HPLC, lipolytic activity in isolated rabbit fat cells, and radioimmunoassay.

NH2-terminal amino acid sequence and peptide mapping of purified human beta-lipotropin: comparison with previously proposed sequences.

Beta-Lipotropin was purified from human pituitary glands to a purity of greater than 90%. The amino acid compositions of beta-lipotropin and its three cyanogen bromide cleavage peptide fragments were in agreement with the structure proposed by Li and Chung [Li, C.H. & Chung, D. (1981) Int. J. Pept. Protein Res. 17, 131-142]. However, the amino acid sequence of its NH2-terminal 46 amino acid residues established here differs both from the sequence derived from the direct sequence analysis of the peptide reported by Li and Chung and from that predicted on the basis of the nucleotide sequence of the human pro-opiolipomelanocortin gene proposed by Chang et al. [Chang, A.C.Y., Cochet, M. & Cohen, S.W. (1980) Proc. Natl. Acad. Sci. USA 77,4890-4894] but agrees with the structure recently derived by direct sequence analysis by Hsi et al. [Hsi, K.L., Seidah, N.G., Lu, C.L. & Chrétien, M. (1981) Biochem. Biophys. Res. Commun. 103, 1329-1335] and predicted on the basis of nucleotide sequence analysis by Takahashi et al. [Takahashi, H., Teranishi, Y., Nakanishi, S. & Numa, S. (1981) FEBS Lett. 135, 97-102]. These discrepancies, found from residues 9 to 25 of beta-lipotropin, could result from pro-opiolipomelanocortin gene polymorphism, from the existence of multiple genes for pro-opiolipomelanocortin, or, more probably, from minor errors in nucleotide and amino acid sequence analyses.

Synthesis and properties of human gamma-lipotropin.

The synthesis of human gamma-lipotropin by the solid-phase method is described. The synthetic product was characterized by Rf in partition chromatography on Sephadex G-50, paper electrophoresis, polyacrylamide gel electrophoresis, thin-layer chromatography, HPLC, end group determination, peptide mapping of a tryptic digest, and amino acid analyses of acid and enzymic digests. The synthetic material is identical to natural human gamma-lipotropin when assayed against natural human beta-lipotropin for lipolytic activity.

Effect of beta-lipotropin on aldosterone production in the isolated rat adrenal cell preparation.

Stimulation of aldosterone and corticosterone production by pituitary peptides structurally or biosynthetically related to ACTH was investigated in suspensions of isolated rat adrenal glomerulosa and fasciculata cells, respectively. Three different preparations of highly purified ovine (Li) or human (Chrétien and Orth) beta-lipotropin (beta LPH) were tested. In contrast to synthetic ACTH-(1-24), which stimulated aldosterone secretion at concentrations of 10(-12)-10(-11) M, beta LPH concentrations of 10(-8)-10(-6) M were required for significant stimulation. Stimulation of corticosterone production by beta LPH preparations generally paralleled their aldosterone-stimulating activity, and most steroidogenic activity could be accounted for by immunoreactive ACTH, as determined in two ACTH RIAs. Synthetic human beta LPH-(37-58), which contains the 47-53 heptapeptide sequence common to beta LPH and ACTH, had aldosterone- and corticosterone-stimulating activities similar to those of equimolar concentrations of beta LPH, whereas synthetic fragments of the COOH-terminal (61-91) portion of beta LPH had no steroidogenic activity. These data indicate that part of the slight steroidogenic activity of purified beta LPH preparations is due to contaminating ACTH, and part is due to the intrinsic ACTH-like activity conferred upon beta LPH by the amino acid sequence shared with ACTH. In contrast to ACTH, the concentrations of beta LPH required to stimulate adrenal steroidogenesis were 10(2)-10(5) times greater than normal plasma levels, indicating that physiological pituitary beta LPH secretion has no direct role in regulating the secretion of aldosterone.

The control of steroidogenesis by human fetal adrenal cells in tissue culture. III. The effects of various hormonal peptides.

The effects upon production of cortisol and dehydroepiandrosterone (DHA) by human fetal adrenal cells in tissue culture were studied using commercial hCG (0.5 and 5 IU/ml), purified hCG (0.7-6.7 IU/ml), the alpha-subunit of hCG (200 and 1000 ng/ml), human GH (50 and 200 ng/ml), human PRL (0.1-100 ng/ml), alpha-MSH (0.1-10 ng/ml), corticotropin-like intermediate lobe peptide (200 ng/ml), human beta-lipotropin (0.1 and 0.2 ng/ml), and beta-endorphin (100 ng/ml). Although each peptide was added to the culture medium in a concentration either similar to that observed in the fetal circulation or (where such information was not available) in amounts several times greater than those effective for ACTH in this system, none demonstrated any significant stimulation of steroid production. In particular, repeated studies with hCG showed that this hormone had no stimulating effect upon DHA production, neither in cultures of whole adrenals nor in cultures of separated fetal zone and definitive zone cells. Furthermore, none of these peptides showed a synergistic effect upon DHA production when they were added to cultures together with concentrations of alpha-ACTH-(1-24) (10(2)-10(3) pg/ml) previously demonstrated to represent the middle of the dose-response curve. Indeed, the only significant interactions with alpha-ACTH-(1-24) observed in these studies were a slight reduction in cortisol production produced by corticotropin-like intermediate lobe peptide and apparent inhibition of DHA production by beta-lipotropin and GH. The data do not lend credence to the suggestion that any of these peptides plays an important role in vivo in stimulating fetal adrenal steroidogenesis.

Intrinsic analgesic activity of human beta lipotropin in rats: Comparison with other opioid peptides

The intracerebroventricular administration of human beta lipotropin (β h-LPH) prepared from pituitary extracts increased the threshold for vocalization and vocalization afterdischarge in the rat after electrical stimulation of the tail. This effect was of quick onset, of longer duration than that of other opioid peptides, and was naloxone-reversible. On a molar basis, β h-LPH was a more potent analgesic than met-enkephalin but much less potent than the synthetic pentapeptide analogue (D-Ala 2)-metenkephalinaroide or human β-endorphin. Bacitracin, a peptidase inhibitor, markedly potentiated and prolonged the analgesia elicited by any of the peptides used. Furthermore, the analgesia induced by β h-LPH was blocked by the concomitant intraventricular injection of its specific antibody but not by the antibodies against met-enkephalin or human β-endorphin. These findings suggest that the analgesic effect of β h-LPH depends on the intact 91 amino acid peptide and not on any of its biodegradation products.

A simplified radioimmunological method for the determination of human beta-endorphin in cerebrospinal fluid.

Human beta-endorphin-like immunoreactive substances (beta h-EI) in human cerebrospinal fluid (CSF) were determined radioimmunologically. The cross reactivity of the antibodies to human beta-endorphin (beta h-E) amounted to 40% for human beta-lipotropin (beta h-LPH) whilst it was less than 1% for leu- and metenkephalin, alpha- and gamma-endorphin, fraction I and II [5], substance P and alpha-MSH. Prior to radioimmunological determination, an adsorbtion of beta h-EI from CSF with silicic acid was carried out and followed by a desorbtion, using a mixture of aceton/hydrochloric acid. This method was chosen because of the ratio of beta h-LPH to beta h-E in the desorbate can be shifted in favour of beta h-E owing to the variation in recoveries r (r beta h-LPH = 33%, r beta h-E = 64%). On the one hand, this enables a more specific determination of beta h-E and, on the other hand, and separation of any peptidase than may be present [9]. An adsorbtion/desorbtion of 2 ml CSF surfaces to prove the presence of 20-150 pg/ml (65-48 fmol/ml) of beta h-EI. The CSF of 28 patients with various neurological diseases was examined and 24 of them had concentrations of 20-70 pg/ml beta h-EI. The remaining four which had concentrations less than 20 pg/ml, came from meningitis patients undergoing corticoid therapy. A purchasable RIA kid was tested for its determination of beta h-E and was found to be unsuitable.

Action of cathepsin D on human beta-lipotropin: a possible source of human "beta-melanotropin".

Highly purified calf brain cathepsin D (EC 3.4.23.5) selectively splits the Leu77-Phe78 and Ala36-Ala37 peptide bonds of human beta-lipotropin. It is suggested that the formation of human "beta-melanotropin" from gamma-lipotropin, and that of gamma-endorphin from beta-endorphin, is due to the action of cathepsin D during isolation procedures.

Human corticotropin (ACTH) radioimmunoassay with synthetic 1--24 ACTH.

A corticotropin antiserum was obtained from rabbits immunized with synthetic 1--24 corticotropin conjugated with bovine serum albumin. The antiserum did not cross react with synthetic alpha-melanotropin or with synthetic beta-endorphin and had a cross reactivity of 0.23% with human beta-lipotropin. We developed a radioimmunoassay with the antiserum obtained, in which we used polyethylene glycol in conjunction with a second precipitating antibody for fast (15-min) separation of antibody-bound and free corticotropin. The assay had a sensitivity of 16 ng/L and was validated on patients with various pituitary and adrenal diseases. From 103 normal subjects, the median value for corticotropin in specimens collected during the morning was 34 ng/L of plasma; the upper 95% confidence limit of the normal range was 98 ng/L.

MEASUREMENT OF β-ENDORPHIN IN HUMAN PLASMA

beta-endorphin has been identified in human plasma by means of gel filtration and a sensitive radioimmunoassay for human beta-endorphin (beta h-endorphin). Mean baseline plasma beta h-endorphin concentration in 5 individuals was 21 +/- 7.3 (SD) pg/ml (6.2 +/- 2.2 (SD) fmole/ml). Following metyrapone stimulation mean plasma concentration increased to 55.4 +/- 10.1 (SD) pg/ml (16.3 +/- 3.1 (SD) fmole/ml). The molar ratio of human beta-lipotropin (beta h-LPH) to beta h-endorphin was 2.2 in baseline plasma and 2.4 after metyrapone stimulation.