Synthetic Human ACTH Research Articles

Enzyme Immunoassay of Adrenocorticotropic Hormone-like Immunoreactive Substance in Human Plasma.

A sensitive and specific double-antibody enzyme immunoassay (EIA) for adrenocorticotropic hormone-like immunoreactive substances (ACTH-ISs) was developed. In competitive reactions, the ACTH (1-24)-antibody was incubated with both ACTH (1-24) standard (or sample) and 13-D-galactosidase labeled synthetic human ACTH (1-24) (delayed addition). The free and antibodybound enzyme haptens were separated by using an anti-rabbit IgG coated immunoplate. The activity of the enzyme on the plate was fluorometrically determined. The present immunoassay allows for the detection of 2 to 600 fmol/mL of ACTH (1-24). Using the proposed EIA, ACTH (1-24)-ISs in human plasma were thus determined. As a result, the levels of ACTH (1-24)-ISs in human plasma were found to be about 3.7 fmol/mL.

Interrenal activity in the female lizard Lagerta vivipara J.: In vitro response to ACTH 1–39 and to [SAR 1, VAL 5] angiotensin II (ANG II)

A perifusion system technique was developed in order to determine in virto the respective roles of ACTH and ANG II in the regulation of adrenal steroidogenesis in the lizard Lacerta vivipara. Synthetic human ACTH 1–39, administered as 20-min pulses, stimulated corticosterone (B) and aldosterone (A) release in a dose-dependent manner. The increase in corticosterone output was higher than that in aldosterone output, leading to an enhancement of the B/A ratio. Iterative stimulations with 1 nM ACTH (20-min pulses every 120 min) led to reproducible increases in corticosterone and aldosterone release. Prolonged stimulation with 1 nM ACTH (up to 240 min) caused a sustained increase in corticosteroid release, suggesting that, in the lizard, ACTH does not induce any desensitization phenomenon. The angiotensin II analogue [Sar 1, Val 5] ANG II also stimulated corticosterone and aldosterone release in a dose-dependent manner; the stimulatory effects of ANG II on both steroids were very similar. These results indicate that, in lizards, ACTH plays a major role in the regulation of adrenal steroidogenesis. Since ANG II stimulates the production of gluco- and mineralocorticoids, our data raise the question of the existence of two cell types synthesizing corticosterone and aldosterone, respectively, in reptiles.

In vitro study of frog ( Rana ridibunda pallas) interrenal function by use of a simplified perifusion system VIII. Structure-activity relationship of synthetic ACTH fragments and γ-MSH

The present study was undertaken to determine the structure-activity relationships of ACTH analogs on corticosteroid production by frog adrenal gland. Rana ridibunda interrenal dice were perifused with amphibian culture medium for 10 hr. Corticosterone and aldosterone concentrations were measured in the effluent perifusate using sensitive and specific radioimmunoassay methods. Perifusion of interrenal fragments with increasing concentrations of synthetic human ACTH 1–39 (ranging from 6.25 × 10 −11 to 10 −9 M) led to a linear log-dose increase in both corticosterone and aldosterone secretion. Thus, this model made it possible to compare the steroidogenic potency of several ACTH analogs. Synthetic α-MSH and its des-Nα-acetyl derivative were found to be approximately equipotent, and 5 × 10 3 times less active than authentic ACTH. The short-chain analog ACTH 1–10 was 2 × 10 4 times less potent than ACTH whereas ACTH 4–10 was totally inactive. A fragment of the N-terminal region of the proopiomelanocortin molecule, γ 3-MSH, caused a dose-related stimulation of steroid secretion. However, in contrast to what has been observed in the rat, γ 3-MSH did not potentiate the corticotropic action of ACTH on frog interrenal gland. Since processing of proopiomelanocortin in frog intermediate lobe generates high amounts of α-MSH and des-Nα-acetyl α-MSH, these results suggest that in amphibians, several peptides other than ACTH may be involved in the control of corticosteroidogenesis.

Adrenocorticotrophic hormone in the anterior and neurointermediate lobes of the rat during the perinatal period: polymorphism, biological and immunological activities of ACTH.

When acid extracts (0.1 N HCl) of the anterior or neurointermediate lobes of fetal and newborn rat pituitary were subjected to gel filtration on Sephadex G-50 fine columns, polymorphism of ACTH was revealed by radioimmunoassay. At every stage of pregnancy and after birth three main ACTH forms were observed. The first form ('big' ACTH) eluted near the void volume of the column; its apparent molecular weight was 45,000. The second form ('little' ACTH) coeluted with synthetic human ACTH1-39 and 125I human ACTH. As the latter marker eluted more slowly than the former, 'little' ACTH, which showed an apparent molecular weight near 5,000, could not be a single entity. The third form ('intermediate' ACTH) eluted between 'big' and 'little' ACTH; its apparent molecular weight was near 13,000. During the perinatal period, the relative proportions between these different molecular forms of ACTH changed in both anterior and neurointermediate lobes. Thus the gradual increase of 'intermediate' and 'little' forms was accompanied by a decrease of 'big' ACTH. The perfusion of fetal adrenal glands with the same immunological quantities of 'big', 'intermediate' and 'little' ACTH showed that the 'little' and 'intermediate' forms have greater biological potency in eliciting corticosterone secretion than the 'big' ACTH form.

Novel variants of adrenocorticotrophic hormone in porcine anterior pituitary

Extracts of porcine anterior pituitary contain several corticotrophic variants of ACTH 1–39. They were isolated by adsorption chromatography, ion-exchange chromatography and reverse-phase high-performance liquid chromatography. Four variants were then identified as starting and ending at positions corresponding to ACTH 1–38, 1–37, 7–39 and 7–38. Several of these fragments were recovered in chromatographically multiple forms. Although all fragments isolated had corticotrophic activity (measured on isolated rat adrenal cells) those with a shortened N-terminal region had a lower potency than those with an intact N-terminal region. Corticotrophic activity of porcine ACTH 7–38 was detected in a preparation with a β-aspartyl shift at position 25. However, in agreement with previous studies, synthetic human ACTH 7–38, in which an intact Asn-Gly bond was structurally proven, possessed no such activity. The results indicate that position 25 as well as positions 1–6 are important for corticotrophic activity, and that a deamidative β-aspartyl shift at position 25 can influence the activity.

Impaired clearance of beta-lipotropin in uremia.

Immunoreactive ACTH and beta-lipotropin (beta-LPH) plasma concentrations are elevated in clinically stable chronic renal failure patients on hemodialysis (LPH: patients, 271.8 +/- 35.7 fmol ml-1; normal subjects; 6.6 +/- 0.5; ACTH: patients, 56.4 +/- 15.3; normal subjects, 19.4 +/- 1.7). To begin to study the etiology of such elevated levels, the MCR, apparent volume of distribution, and fractional rate of disappearance of synthetic human ACTH and highly purified human beta-LPH were determined in two clinically stable chronic renal failure patients on hemodialysis, after bolus simultaneous injection of both peptides. Biphasic disappearance curves were obtained for beta-LPH; triphasic for ACTH. The MCR of ACTH was within the range seen in normal subjects, whereas the MCR of beta-LPH was less than one half the normal rate. The data indicate that a decrease in MCR (rather than an increase in pituitary secretory rate) may account for the higher plasma levels of beta-LPH in uremic patients.

Synthesis of a corticotropin analog that retains full biological activity after iodination.

In order to circumvent the drastic decrease in biological activity that accompanies iodination of corticotropin, we have synthesized an analog of the human hormone containing phenylalanine in place of tyrosine in position 2 and norleucine in place of methionine in position 4 to give [Phe2,Nle4]ACTH-(1-38). This analog, referred to as Phe2,Nle4-ACTH-(1-38), was purified by ion exchange chromatography and partition chromatography. Phe2,Nle4-ACTH-(1-38) was found to be indistinguishable from synthetic human ACTH in its ability to stimulate corticosterone production in isolated rat adrenocortical cells. Iodination of Phe2,Nle4-ACTH-(1-38) resulted in the formation of monoiodo-Tyr23,Phe2,Nle4-ACTH-(1-38) and diiodo-Tyr23,Phe2,Nle4-ACTH-(1-38), which were completely separated from each other and unmodified Phe2,Nle4-ACTH-(1-38) by reverse phase high performance liquid chromatography. Monoiodo-Tyr23,Phe4,Nle4-ACTH-(1-38) was also found to be as potent as ACTH in stimulating steroidogenesis. These results show that Phe2,Nle4-ACTH-(1-38) can be used for the preparation of the radioligand with full biological activity for studying physiologically relevant corticotropin receptors and for use in RIA.

Forms and activity of high molecular weight adrenocorticotropin in guinea pig

High molecular weight forms of adrenocorticotropin (ACTH) and endorphin were identified in extracts of guinea pig anterior and intermediate/posterior pituitary. Extracts of anterior pituitary contained ACTH immunoactive material with apparent molecular weights of 36,000, 24,000 and 4,500 daltons. The highest molecular weight form of ACTH co-migrated with a peak of endorphin immunoactive material. No material the size of glycosylated ACTH(1–39) was detected. Separated forms of high molecular weight ACTH prepared from mouse tumor cell culture medium stimulated the same maximal production of steroid as ACTH(1–39) in the guinea pig adrenal cell bioassay. Pro-ACTH/endorphin and ACTH biosynthetic intermediate were two orders of magnitude less potent than synthetic human ACTH(1–39); glycosylated ACTH(1–39) was equipotent to ACTH(1–39) although no similar material was detected in guinea pig pituitary extracts. Isolated guinea pig adrenal cortical cells were incubated with the various separated forms of mouse tumor cell ACTH and products synthesized from ( 3H)pregnenolone were analyzed by two-dimensional thin-layer chromatography. The ratio of cortisol-related to corticosterone-related products was the same in response in glycosylated and nonglycosylated ACTH.

Analysis of Human Pituitary and Tumor Adrenocorticotropin Using Isoelectric Focusing*

ABSTRACT Heterogeneity of immunoreactive ACTH was investigated in extracts of three human pituitary glands, three tumors obtained from patients with the ectopic ACTH syndrome, and a porcine pituitary gland using gel filtration and isoelectric focusing. A single gel filtration fraction of immunoreactive ACTH was applied to isoelectric focusing and pis were measured using RIAs specific for the amino-terminal portion (N-ACTH) and for the carboxyl-terminal (C-ACTH) of ACTH. The little ACTH peak of the human pituitary extract separated on gel filtration was found to contain four components with different pis on isoelectric focusing. The distribution of these peaks was almost identical in each pituitary extract, and the major peak in each case showed a pi value of 8.0, a value similar to that of synthetic human ACTH. The little ACTH peak of the tumor extract was composed of four or five peaks with different pi values. Each peak of the tumor extracts corresponded to some peak found in the pituitary extracts, and...

An Adrenocorticotropin Analog [ACTH(6-39)] which Acts as a Potent in Vitro Adrenocorticotropin Antagonist at Low Calcium Concentration and as a Weak Agonist at High Calcium Concentration*

We have characterized in two different in vitro assay systems the biological activity of a synthetic human ACTH analog containing residues 6–39 of the native sequence. In broken membranes from rat adrenal cortex, the analog did not stimulate adenylate cyclase but blocked the stimulatory effects of ACTH(l–24) at equimolar concentrations. In this system, the Kd (Kact) for ACTH(l–24) was approximately 0.41 μM, while the Ki of ACTH (6–39) was 0.32 –M. The analog showed no agonist effects in the membrane system over a wide range of calcium concentrations but, in the presence of the GTP analog, Gpp(NH)p, became a very weak agonist. In a second assay system utilizing dispersed intact rat adrenal cells, the analog was found to inhibit both steroidogenesis and cAMP production in response to ACTH(1–24). In the intact cell system, a 50-to 100-fold molar excess of inhibitor was required to block steroidogenesis. The apparent Kd of ACTH(l–24) for steroidogenesis was approximately 0.1 nM, while the Ki of the antagonist...

Comparative metabolic clearance rate, volume of distribution and plasma half-life of human β-lipotropin and acth

An antiserum to β h-lipotropin (LPH) which does not cross react with β h-endorphin has been obtained utilizing two different methods of affinity chromatography. This was employed in studies of three normal human subjects in whom the metabolic clearance rate (MCR) apparent volume of distribution (V d) and fractional rate of disappearance (K d) of ACTH and β h-LPH were determined following bolus simultaneous injection of 270 μg highly purified β h-LPH and 230 μg of synthetic human ACTH. A biphasic disappearance curve was noted for both hormones. β h-LPH : MCR-0.571, 0.519, and 0.461 L/minute; V d-30.7, 27.7, 25.0 liters, representing 49, 46 and 35% of body weight; K d-0.0186, 0.0187, 0.0185 min −1. ACTH : MCR-0.274, 0.266, and 0.332 L/minute; V d-6.5, 6.5, 14.5 liters, representing 10.4, 10.8 and 20.4% of body weight; K d-0.0418, 0.0409, 0.0229 min −1. The observed larger MCR of β h-LPH can account for previous observations of basal plasma ACTH/LPH ratios greater than unity, even though these peptides are present in the pituitary in equimolar concentrations.

ChemInform Abstract: SYNTHESIS OF CORTICOTROPIN PEPTIDES. X. THE SYNTHESIS OF ACTH(1‐18)‐OH, ACTH(1‐18)‐NH2 AND ACTH(1‐19)‐NH2

AbstractDie Synthese der Oktadekapeptide (Ia) und (Ib), die den Aminosäuresequenzen 1‐18 von Corticotropin (ACTH) entsprechen, erfolgt durch Kupplung des geschützten N‐Hydroxysuccinimidesters des ‐ bekannten ‐ N‐terminalen Dekapeptids mit den ‐ ebenfalls bekannten ‐ entsprechenden Oktapeptidderivaten, Schutzgruppenabspaltung und Reinigung.

Synthesis of Corticotropin Peptides. XIII. The Synthesis of a Hexacosapeptide and a Heptacosapeptide Corresponding to the First Twenty-six and Twenty-seven Amino Acid Residues of Corticotropin (ACTH)

Abstract The syntheses are described of a hexacosapeptide H–Ser–Tyr–Ser–Met–Glu–His–Phe–Arg–Trp–Gly–Lys–Pro–Val–Gly–Lys–Lys–Arg–Arg–Pro–Val–Lys–Val–Tyr–Pro–Asn–Gly–OH (I) and a heptacosapeptide H–Ser–Tyr–Ser–Met–Glu–His–Phe–Arg–Trp–Gly–Lys–Pro–Val–Gly–Lys–Lys–Arg–Arg–Pro–Val–Lys–Val–Tyr–Pro–Asn–Gly–Ala–OH (II) corresponding to the first twenty-six and twenty-seven amino acid residues, respectively, of corticotropin (ACTH), in which a new protecting group 1-methylcyclohexyloxycarbonyl (Mhoc) is utilized for the temporary protection of the ε-amino function of lysines. The in vivo steroidogenic potencies of the synthetic peptides I and II are compared on a molar basis with those of ACTH(1–18)-NH2 and synthetic human ACTH (αh-ACTH) to demonstrate that the relative potencies of I and II vs. ACTH(1–18)–NH2 are 2.3 and 2.3–3.8, respectively, and those vs. αh-ACTH are 1.1 and 1.1–1.8, respectively.

Synthesis of Corticotropin Peptides. X. The Synthesis of ACTH(1–18)–OH, ACTH(1–18)–NH2 and ACTH(1–19)–NH2

Abstract The final coupling, deprotection and purification processes for the synthesis of octadecapeptides, H–Ser–Tyr–Ser–Met–Glu–His–Phe–Arg–Trp–Gly–Lys–Pro–Val–Gly–Lys–Lys–Arg–Arg–OH(I) and H–Ser–Tyr–Ser–Met–Glu–His–Phe–Arg–Trp–Gly–Lys–Pro–Val–Gly–Lys–Lys–Arg–Arg–NH2 (II), corresponding to the first eighteen amino acid residues of corticotropin (ACTH) are described, in which the N-hydroxysuccinimide ester of the N-terminal decapeptide derivative is coupled with the C-terminal octapeptide and octapeptide amide derivatives to give the two protected end products. The same decapeptide active ester is also utilized to the synthesis of a nonadecapeptide, H–Ser–Tyr–Ser–Met–Glu–His–Phe–Arg–Trp–Gly–Lys–Pro–Val–Gly–Lys–Lys–Arg–Arg–Pro–NH2 (III), identical with the first nineteen amino acid residues of ACTH. The in vivo steroidogenic potencies of the synthetic peptides are compared with that of the synthetic human ACTH (αh-ACTH) as reference to show that the relative potencies of I, II, and III vs. αh-ACTH are 0.14, 0.48, and 0.47, respectively, as estimated on a molar basis.

Method for Preparation of Ampoules Containing Microgram Quantities of Polypeptide Hormones for Distribution and Use as Laboratory Standards: Synthetic Human ACTH andβ-MSH1

There are a number of polypeptide hormones, their subunits, and other derivatives that are available in such short supply as to preclude distribution by conventional methods to the many qualified investigators who require them. This report describes a method for preparing sealed ampoules of two such hormones, the utilization of which can provide many qualified investigators with comparable, stable reference compounds for several years and eliminate unnecessary waste and error associated with the distribution and use of bulk quantitites of peptide hormone preparations. Sealed ampoules of synthetic human adrenocorticotrophic hormone (alpha(h)(1-39) ACTH) and beta melanocyte-stimulating hormone (beta(h)(1-22) MSH) were prepared. Each ampuole contains 50 mug of dried and recoverable peptide plus 1 mg of lactose, sealed under dry nitrogen at atmospheric pressure. The respective preparations are fully active, both biologically and radioimmunologically, and are suitable as reference compounds in all types of biologic and radioimmunologic assays of these hormones. In addition, radioactive iodine labeling of each peptide can be accomplished without prior removal of the lactose.

Treatment of a case of allergic asthma with a synthetic human ACTH preparation

The following case history documents the first patient treated with synthetic human ACTH. It was prescribed for an asthmatic child because of suspected allergy to both porcine ACTH and tetracosactrin. It proved both effective clinically and free of any adverse effects.

Prolonged Corticotropic Action of Synthetic Human ACTH in Man

The corticotropic activities of synthetic human ACTH (revised structure), synthetic porcine ACTH (unrevised structure) and synthetic 1-24ACTH were compared in 8 normal men by measuring the magnitude and duration of the elevation of plasma 11-hydroxy-corticosteroids following the injection of 1 mg of the peptides. The duration of response was significantly longer after human ACTH than following porcine or 1-24ACTH, although the initial responses were identical. This difference was noted after both intramuscular and intravenous administration. The data suggest that the carboxyterminal of the ACTH peptide is of some importance for biological activity and that biological species specificity may exist.

LEVELS OF CORTICOTROPHIN ANALOGUES IN THE BLOOD AFTER INFUSION INTO RATS

An isolated rat adrenal cell bioassay was used to measure blood concentrations in rats after infusion of synthetic human ACTH, corticotrophin-(1-24)-tetracosapeptide or [D-Ser1, Lys17, Lys18]corticotrophin-(1-18)-octadecapeptide amide. Lower blood levels were found with the 1-24 peptide than with human ACTH and the highest levels were found with the 1-18 peptide. These results suggest that the 1-24 peptide which is almost equipotent with natural ACTH in vivo may be more potent at the receptor and corroborate findings to this effect obtained with isolated adrenal cells. The high potency and prolonged action of the 1-18 analogue in vivo are also explained by these results. Low arterial blood concentrations of the 1-24 peptide and human ACTH were found during infusion, suggesting that substantial inactivation must be occurring in a single passage through the lungs. The effects of renal ligature on blood concentrations indicated that the kidney is involved in handling the 1-18 peptide and that human ACTH is also cleared by this organ. After infusion the fall in blood concentrations was biphasic. It is suggested that the rapid phase is due to clearance of peptides in the circulation which results in a fall to lower blood concentrations which are sustained by slow release of peptide from binding sites which act as a depot.

A radioimmunoassay for plasma corticotropin in frogs ( Rana esculenta L.)

A radioimmunoassay technique has been developed for measuring frog plasma corticotropin (ACTH) without prior extraction. Using synthetic porcine ACTH as a reference standard, 131I-labeled synthetic human ACTH (sp act > 500 mCi/mg) as tracer and rabbit anti-porcine ACTH serum, the lower measurable value was estimated at about 4 pg ACTH. Only human and porcine ACTH, 1–24ACTH, and frog pituitary ACTH reacted with the rabbit anti-porcine ACTH serum. No cross-reactivity has been found with synthetic 1–16,17–39ACTH, αMSH, and bovine βMSH. Appearance of damaged 131I-h ACTH components after storage in plasma solutions was followed for 7 days. The conditions making it possible to reduce ACTH damage have been ascertained. The average plasma corticotropin level (±CI) was found to be 38.8 ± 7.8 pg/ml without any significant difference between males and females. These results suggest that frog ACTH secretion has much in common with mammalian secretions.

IMMUNOREACTIVE ACTH AND CORTISOL PLASMA LEVELS DURING PREGNANCY. DETECTION AND PARTIAL PURIFICATION OF CORTICOTROPHIN‐LIKE PLACENTAL HORMONE: THE HUMAN CHORIONIC CORTICOTROPIN (HCC)

The high plasma cortisol and ACTH levels present in pregnant women as well as the non-parrallelism of their plasma extract dilution curves in comparison with the standard curve in the ACTH radioimmunoassay, are evidence for the presence of an ACTH-like substance during pregnancy which would interfere with the assay. Placental extracts were obtained by acid-acetone extraction, followed by partial purification with oxycellulose and by extraction with porous glass powder. A substance was detected which partially cross-reacted with synthetic human ACTH in the radioimmunoassay and which showed biological activity using the assay procedure described by Liscomb & Nelson. The data sustain the existence of an ACTH-like placental hormone: human chorionic corticotrophin (HCC).