Human Growth Hormone Preparations Research Articles

Inhibition of recombinant human growth hormone-induced and prolactin-induced activation of neutrophils by octreotide.

Growth hormone, prolactin and somatostatin are polypeptide hormones of the neuroendocrine and peripheral nervous systems. In vitro, these have opposing effects on cells of the immune system. We compared the effects of these peptides on activation of neutrophils using a recombinant preparation of human growth hormone, human prolactin and octreotide, a long acting analog of somatostatin. In the absence of growth hormone, octreotide did not affect either neutrophil locomotion or respiratory burst. Octreotide, however, significantly antagonized growth hormone-induced activation of neutrophils for enhanced respiratory burst as well as growth hormone-induced inhibition of stimulated migration. As the effect of growth hormone on neutrophils is mediated by the prolactin receptor, its inhibition by octreotide was also tested using prolactin as priming agent. Data indicate comparable effects of octreotide on priming of neutrophils by prolactin. The effect of octreotide was dose-dependent and appeared to be selective, as activation of neutrophil respiration burst by gamma-interferon, and inhibition of stimulated migration by tumor necrosis factor-alpha were unaffected by octreotide. The present study suggests that octreotide may act on neutrophils directly by antagonizing growth hormone or prolactin at the cellular level.

Effect of somatotropin of mammalian cell origin in growth hormone deficiency.

Sixty-nine growth hormone-deficient patients were treated for 1 year with somatotropin (recombinant DNA-derived human growth hormone) produced in mouse cells. The growth velocity of the 50 patients (72%) in whom the effectiveness of this growth hormone could be evaluated increased from a mean (+/- SD) of 3.5 +/- 1.1 to 8.7 +/- 1.6. cm/y. An enhanced rate of weight gain was also observed. Bone age was not unduly accelerated. One of 66 patients developed antibodies to recombinant growth hormone, which did not affect the response to therapy. No patient developed antibodies to host cell proteins. An increased insulin response to a standard glucose load, without any change in glucose tolerance, was observed after 1 year of treatment. This authentic sequence human growth hormone preparation produced in mammalian cells is both effective and safe in the treatment of children with growth hormone deficiency.

Immunogenicity of a mammalian cell-derived recombinant human growth hormone preparation during long-term treatment.

The development of anti-human growth hormone (anti-hGH) and anti-host-cell protein antibodies to recombinant hGH (rhGH) of mammalian cell origin was determined in 395 children (304 GH deficiency; 91 Turner syndrome) undergoing long-term treatment (up to 54 months) for growth disorders. In all patients, blood samples were obtained prior to and every 2-3 months during treatment, and analyzed at a central laboratory for anti-hGH antibodies by RIA and antibodies to host-cell antigens by ELISA. During the first 24 months of treatment, 9 (3%) of the 304 patients with GH deficiency developed antibodies to rhGH for longer than 3 months. However, persistent antibodies were seen in only 2 patients, both of whom had proven hGH-N gene defects. In the remaining 7 (2%) anti-rhGH-antibody-positive patients, antibody concentrations showed a tendency to increase for 3-12 months, irrespective of the time of onset of measurable concentrations, and declined thereafter. In these patients, binding capacities were between 0.01 and 0.1 mg/l, and binding affinities were between 7 x 10(8) and 8 x 10(9) l/mol. Height velocity was unaffected in these children. None of the 91 patients with Turner syndrome developed persistent anti-hGH antibodies. Further, no child developed antibodies to host-cell antigens during treatment with rhGH of mammalian cell origin.

Analysis of human pictuitary growth hormone and its charge varriants by fast‐atom bombardment mass spectrometry

There is evidence that even highly purified preparations of human growth hormone are not homogenous, but contain charge as well as size variants. The charge heterogeneity was suggested to be due to deamidation of the native hormone. To verify this we have applied peptide mapping followed by fast-atom bombardment mass spectrometry (FAB-MS), in order to identify fragments containing the altered amino acids. Growth hormone was purified from human pituitaries and the differently charged forms were separated by column electrophoresis in agarose suspension. The isolated components were treated with trypsin and analysed directly by FAB-MS without prior separation by reversed-phase high-performance liquid chromatography (RP-HPLC). Using this technique, approximately 80% of the hormone structure was recovered and two deamidation sites were found in the fragment T15 (FDTNSHNDDALLK). The results clearly elucidated the potential use of FAB-MS for the fast screening of other variants of the growth hormone which are known to exist.

Comparative Study of Biosynthetic Human Growth Hormone Immunogenicity in Growth Hormone Deficient Children

The immunogenicities of six recombinant human growth hormone (rhGH) preparations, from KABI (A rhGH191 and B rhGH192), Eli Lilly (C), Nordisk (D), Sanofi (E) and Serono (F), used to treat 260 GH-deficient children, have been compared using a common specific and sensitive procedure for antibody determination. For this purpose we developed two immunoassays: a competitive liquid radioimmunoassay using 125I-rhGH, and an immunometric solid enzymoimmunoassay in which the rhGHs were immobilized. Blood samples were collected from the GH-deficient children before treatment and after 3, 6, 9, 12, 18 and 24 months of therapy. Human GH antibodies were detected in children treated with 3 of the 6 rhGH preparations. Seven percent of the patients treated with hormone A, 14% with hormone B and 22% with hormone C formed antibodies against the respective rhGH. Differences in capacity and affinity of the hGH antibodies were observed between these anti-GH-positive groups. They could be divided into 2 groups according to their immunopotency. One group (7, 14 and 6% of the patients treated with hormones A, B and C, respectively) developed anti-hGH antibodies with very low binding capacities (30-100 fmol/ml). The other group (16% of the patients treated with hormone C) developed IgG-type antibodies to hGH with higher binding capacities (200-1,200 fmol/ml) and a measurable binding affinity (Ka = 10(8) M-1). These hGH antibodies partially inhibited the binding of labeled GH to its specific liver membrane receptor. However, because of their low titer, they did not inhibit growth in the treated children.(ABSTRACT TRUNCATED AT 250 WORDS)

Application of high-performance hydrophobic-interaction chromatography to the characterization of recombinant DNA-derived human growth hormone

This publication analyzes different preparations of recombinant human growth hormone (rhGH) by hydrophobic-interaction chromatography (HIC). The effect of temperature on the separation was investigated as well as a series of commercially available HIC columns (TSK-phenyl-5PW, TSK-ether-PW, Beckman CAA-HIC and polypropyl A). The TSK-ether column gave the best results in the analysis of rhGH samples at different temperatures, as well as allowing an efficient separation of methionyl-hGH from rhGH. The TSK-phenyl column can be effectively used in the examination of different human growth hormone variants. The details of sample preparation have been demonstrated to be important in HIC analysis of hGH on the TSK-ether-5PW column. Injection volume and the solvent used to dissolve the protein sample are both crucial factors in this analysis. Also protein aggregation may play a role in these observations. The effect of temperature, protein concentration and spectroscopic data on the eluted protein suggest, however, that aggregation is not the cause of frontal peaks

Preparation of 125I-labeled human growth hormone of high quality binding properties endowed with long-term stability.

125I-labeled human growth hormone (125I-labeled.hGH) was prepared by using two variants of the chloramine T labelling procedure and purified by polyacrylamide gel electrophoresis (PAGE) of the reaction mixture. Variant A produced a tracer with high specific activity (100 +/- 10 microCi/microgram), high maximal binding capacity to antibodies (93%) and long-term stability (at least 150 days at -20 degrees C). No diiodinated tyrosil residues could be detected in this tracer. Variant B was devised to obtain higher yields of labeled hormone. The electrophoresis of the iodination mixture revealed two radioactive components with Rm values of 0.49 and 0.55 which result from the iodination of hGH variants preexisting in the starting material. Both tracers had similar specific activities (70 +/- 10 microCi/microgram), high maximal binding capacity to antibodies or receptors (80-100%, after 80 days of their obtention) and high stability (at least 100 days at -20 degrees C). It is concluded that the iododerivatives of hGH obtained by either method are adequate to perform radioimmunoassay and receptor studies and have long-term stability.

Inactivation of human immunodeficiency virus during human growth hormone production.

Since human immunodeficiency virus (HIV) has been found in the brains of AIDS patients, the possibility was investigated that preparations of human growth hormone (hGH) extracted from human pituitary glands harbor infectious HIV. It was found that the procedure used for extracting hGH, i.e. a combination of acid pH and 10% ethanol, totally inactivates HIV infectivity.

The effects of biosynthetic insulin-like growth factor-1 supplementation on somatic growth, maturation, and erythropoiesis on the neonatal rat.

Somatomedins are anabolic hormones that may stimulate growth during the perinatal period. To test this hypothesis, neonatal rats were injected with a biosynthetic somatomedin, insulin-like growth factor 1 (IGF-1) twice daily for the first 2 wk of life. Two biosynthetic IGF-1 preparations of different potency were tested as well as a preparation of human growth hormone in five litters of rats. When compared to saline-injected rats, IGF-1 injected rats had increased body weight and tail length as well as specific increases in weights of liver, brain, heart, and testes. In addition, significant increases in bone marrow erythropoietic cell precursors were apparent after IGF-1 injection. IGF-1-treated neonatal rats also exhibited precocious eye opening as a sign of epithelial cell differentiation. Five additional litters of rats received similar injections but were exposed to postnatal nutritional deprivation via artificially increasing litter size. Although IGF-1 caused stimulation of bone marrow erythropoiesis and precocious eye opening, no effects of IGF-1 on somatic or organ growth could be documented. This represents the first demonstration in vivo of the anabolic effects of IGF-1 in rapidly growing neonatal rats but suggests that nutritional sufficiency may also be necessary for the full expression of somatomedin effects.

A rapid method for the preparation of 1251-labelled human growth hormone for receptor studies, using reverse-phase high performance liquid chromatography

Human growth hormone was labelled with 125 Iodine by the stoichiometric modification of the chloramine-T method to a specific activity of 50-80 microCi/microgram, and the iodinated mixture was purified by reverse-phase high performance liquid chromatography using a C18 column (SynChropak RP-P) and a linear gradient. Compared with the usual Sephadex G-100 chromatography, HPLC gave a much better separation, with a higher yield and a considerably reduced analysis time (30 min vs 5 h). The [125I]-labelled preparation had normal binding to IM-9 lymphocyte receptors. The maximum bindability of the HPLC-purified preparation approximated 90%, which is the best value so far reported for human growth hormone. It is concluded that HPLC is a fast, convenient and reproducible method for obtaining an improved [125I]-labelled human growth hormone for receptor studies.

The International Standard for Human Growth Hormone for Bioassay: calibration and characterization by international collaborative study

Two preparations of human growth hormone (hGH) were prepared as candidates for the International Standard for Human Growth Hormone for Bioassay and were studied by 22 laboratories in 10 countries in an international collaborative study. The 2 candidate preparations, freeze-dried in ampoules coded 80 505 and 80 521 , were assayed against the International Standard for Growth Hormone, bovine, for Bioassay (IS bGH), by in vivo assays; against the International Reference Preparation of Growth Hormone, human, for Immunoassay (IRP hGH), by receptor-, immunoassays and other in vitro methods; and against each other by various methods. Both preparations contained the 2 recognized main growth hormone components (22 kDa and 20 kDa forms) and other components, but that in ampoules coded 80 505 had less deamidated hGH and contaminant hormones and pyrogen than that in ampoules coded 80 521 . The estimates by in vivo bioassays, using immature hypophysectomized rats, of the potency of 80 505 in terms of the IS bGH were heterogeneous (1–6 IU/ampoule), probably because of the dissimilarity of the preparations of bovine and human GH. Estimates with receptor- and immunoassays of 80 505 against the IRP hGH were also heterogeneous (3–9 IU/ampoule). Nevertheless, the majority of estimates from all assays tended to be between 3 and 6 IU/ampoule. Although 80 521 and 80 505 differed in relatively minor respects, assays of one directly against the other gave relative potency estimates with in vivo assays which were significantly different from estimates with in vitro methods. With the agreement of the participants in the study, the WHO Expert Committee on Biological Standardization established the preparation in ampoules coded 80 505 as the First International Standard for Human Growth Hormone for Bioassay, with the defined potency of 4.4 IU/ampoule. This corresponds to an approximate 2.5 IU/mg of hGH extract and maintains reasonable continuity of the unit.

Highly purified human growth hormone fails to stimulate lipolysis in rabbit adipocytes in vitro or in rabbits in vivo

To determine whether the rapid lipolytic effect observed with human growth hormone (hGH) preparations in rabbits and rabbit adipose tissue is an intrinsic property of the hormone, we examined the lipolytic effects in vivo and in vitro of clinical grade preparations, hGH purified by DEAE cellulose chromatography, and hGH prepared by recombinant DNA techniques. Using isolated rabbit perirenal adipocytes, ACTH and clinical-grade hGH preparations both stimulated glycerol release to the same maximal rate with half-maximal hGH effects observed between 8 and 50 μg/mL. Purification of the most potent lipolytic preparation of clinical grade hGH by DEAE cellulose chromatography yielded a preparation (2 IU/mg of growth activity that retained insulinlike effects on rat fat pad [U- 14C] glucose metabolism) that, at concentrations up to 0.2 mg/mL, failed to stimulate lipolysis by adipocytes incubated for one or four hours in the presence or absence of dexamethasone or trypsin inhibitor or when preincubated for three hours prior to addition of hGH. Recombinant DNA-derived hGH did not stimulate glycerol release at 0.1 mg/mL. While antiserum to purified hGH blocked the increase in glucose oxidation in rat fat pads produced by clinical grade hGH, it did not inhibit its lipolytic effect using rabbit adipocytes. Purified hGH (0.1 mg/kg IV) was also unable to elicit a rise serum free fatty acid (FFA) levels of conscious rabbits while, at the same dose, clinical grade hGH increased FFA levels to 900 μEq/L over basal. Rapid lipolytic stimulation in rabbit adipocytes by hGH preparations could not be attributed to the 20,000 molecular weight variant of hGH (hGH20K) or the peptide corresponding to positions 32 to 46 of hGH (deletion peptide). Glycerol release was accelerated by a < 5000 molecular weight fraction obtained during the preparation of purified hGH. In conclusion, the lipolytic effect produced by clinical-grade hGH preparations in rabbits in vivo and in vitro could not be attributed to intact hGH, hGH20K, or deletion peptide, but is elicited by a contaminant separable from hGH on further purification.

Fragments of human growth hormone produced by digestion with bromelain: Chemistry and biological properties

In an effort to produce small discrete fragments of human growth hormone (GH), we examine the action of the proteolytic enzyme, bromelain, on this molecule. Purified human GH incubated for 40 min at 22°C with crude bromelain and gel-filtered on Sephadex G-100 resulted in a major digestion product, peak 2. SDS-urea gel electrophoresis in the presence of β-mercaptoethanol suggested that peak 2 was composed of two polypeptide chains. Two polypeptide fractions were isolated by the reduction and S-alkylation of peak 2 in 6 M guanidine-HCl and subsequent chromatography on Sephacryl S-200 in 6 M guanidine-HCl. These two fractions, A and B, had the same mobilities as the two components of peak 2 on SDS-urea gels. Amino-terminal analysis, tryptic peptide mapping, carboxypeptidase digestion, cyanogen bromide cleavage, and amino acid analysis of fraction A indicated that it was peptide 1–135. Amino-terminal analysis and tryptic peptide mapping of fraction B suggested the presence of a mixture of peptides 143–191, 145–191 and 146–191. Thus, peak 2 is heterogeneous and appears to be a mixture consisting of peptide 1–135 + peptide 143–191, peptide 1–135 + peptide 145–191 and peptide 1–135 + peptide 146–191, in each case the N-terminal peptide being joined to the C-terminal peptide by the disulfide bridge between residues 53 and 165. In the weight-gain test in hypophysectomized rats, two preparations of peak 2 appeared to be somewhat less active than the native human GH preparations from which they were derived. Several preparations of peak 2 showed equivalent potency in stimulating [ 14C]glucose oxidation to 14CO 2 by isolated epididymal adipose tissue of hypophysectomized rats. Also, most of the peak 2 preparations were somewhat less active than native human GH in displacing 125I-labeled human GH bound to antibodies to human GH.

Studies of a human growth hormone preparation used for clinical treatment in Great Britain.

This report describes some of the properties of a clinical-grade preparation of human growth hormone (hGH) extracted from acetone-preserved autopsy human pituitary glands and used in Great Britain from 1967 to 1980. Gel filtration of this hGH on Sephadex G-100 yielded (on a weight basis) an average of 48% of a high molecular weight fraction, 10% of an intermediate fraction expected to contain dimeric forms of the hormone and 33% of a fraction considered to be the hGH monomer. The immunoassay potency of the monomer fraction was twice that of the clinical-grade preparation and the amino-acid composition of the monomer fraction agreed well with that obtained from published hGH sequence data. The results of polyacrylamide-gel electrophoresis (under reducing and dissociating conditions) and amino-acid analysis of the high molecular weight fraction suggest that it contains around 30% of aggregated hGH as well as other material not separated from hGH by the purification procedure.

Chondrocyte growth factor from the human pituitary gland.

Extracts of frozen human pituitaries were mitogenic in a fetal rabbit chondrocyte bioassay. In the presence of 10% fetal bovine serum, a 10-fold increase in chondrocyte cell number was observed upon addition of the pituitary factor to the culture medium. After gel filtration, the chondrocyte growth factor eluted with proteins of approximately 40,000 molecular weight. These fractions were pooled and purified further upon ion exchange chromatography using DEAE-cellulose. The most active fraction stimulated cell proliferation in a dose-dependent manner down to 10 ng/ml. The chondrocyte growth factor was trypsin- and heat-sensitive (100 degrees C, 10-15 min). Its isoelectric point (pI 7.9) was different from bovine brain and pituitary fibroblast growth factor (pI 4.8-5.8 and pI 9.5, respectively. Unlike the somatomedins and epidermal growth factor, it was acid-labile. Preparations of human growth hormone, prolactin, luteinizing hormone, and follicle-stimulating hormone, prolactin, luteinizing hormone, and follicle-stimulating hormone, as well as vasopressin and oxytocin, were inactive in the bioassay, demonstrating that the human pituitary contains a chondrocyte growth factor which appears to be distinct from these anterior and posterior pituitary hormones.

Growth Increments With Low Dose Intermittent Growth Hormone and Fluoxymesterone in First Year of Therapy in Hypopituitarism

The availability of a commercial preparation of human growth hormone (hGH) prompted a study in children with hypopituitarism to determine the efficacy of a lower dose of hGH. Thirteen children, ages 4 years 3 months to 13 years 7 months, were given hGH concomitant with fluoxymesterone in an intermittent regimen. The hGH was given intramuscularly, 1 IU daily for 12 to 36 days in each course. There were four courses per year. The fluoxymesterone was given orally, 1 to 2 mg. The total amount of hGH administered was between 48 and 112 IU per year, as compared with the regimen of 312 IU per year, 2 units three times a week. The growth velocity in all 13 children improved, and the mean growth velocity was 6.4 cm +/- 1.13 SD. The mean bone age increment was 0.57 years +/- 0.14 SEM in one chronological year of therapy. The low dose regimen permits a significant reduction in cost of hGH without causing undue advance in bone age.

Effect of growth hormone on vitamin D metabolism.

BEFORE affecting calcium and phosphorus metabolism, vitamin D3 (cholecalciferol) undergoes two hydroxylations; first in the liver, gut and perhaps other organs, to its major circulating form1,2 25(OH)D3 and then in the kidney3 to 1α,25(OH)2D3, the hormonal form. 1α,25(OH)2D3 is the most potent metabolite of cholecalciferol and may be chiefly responsible for all the effects of the vitamin on calcium metabolism4. Although the regulation of the secretion of 1α,25(OH)2D3 by the kidney has been a controversial subject, it is generally agreed that 1α,25(OH)2D3 itself5,6, the calcium and phosphorus content7–9 of the diet and parathyroid hormone (PTH)10 are all involved. But these factors do not seem adequate to account for the increased content of 1α,25(OH)2D3 in the plasma during growth spurts, pregnancy and lactation11, when the major increases in calcium and phosphorus absorption occur. We thought there might be a clue in the action of prolactin, which strongly increases the secretion of 1α,25(OH)2D3 in birds12,13 and enhances plasma 1α,25(OH)2D3 in lactating mammals14. Suppression of prolactin by administration of bromocriptine to lactating rats markedly lowers plasma 1α,25(OH)2D3 levels, although the drug has no such effect in non-lactating controls15. The similarity of the amino acid sequences of prolactin and growth hormone suggested that these hormones might have similar effects. We now report that administration to rats of a highly purified preparation of human growth hormone restores to normal the depressed plasma levels of 1α,25(OH)2D3 produced by hypophysectomy.

Optical activity of disulfide bonds in proteins. 1. Studies on plasmin modified human somatotropin.

The reduction and alkylation of the two disulfide bonds in a preparation of human pituitary growth hormone which had been previously modified by limited proteolysis with the enzyme plasmin have been studied. Quantitative and selective reduction of the carboxyl-terminal disulfide, as well as total reduction of both disulfides, has been achieved in the absence of denaturants. Circular dichroism spectra of the various reduced and reduced-alkylated derivatives have provided sufficient information to allow an estimation of the individual contributions of each disulfide bond to the total optical activity of the protein. These contributions were found to represent a significant portion of the total optical activity between 290 and 250 nm. The carboxyl-termimal bond exhibits negative dichroism with an apparent center near 258 nm ([theta]M,258nm = 2100 deg cm2 dmol-1). By comparison, the contribution of the remaining disulfide is red-shifted to 273 nm, is also negative in sign, and somewhat more intense ([theta]M,273nm = 3200 deg cm2 dmol-1). Circular dichroism measurements have also been used to approximate the rate of reduction of the protein.

REGULATION OF THE ACTIVITIES OF THE ENZYMES INVOLVED IN THE METABOLISM OF STEROID HORMONES IN RAT LIVER: THE EFFECT OF ADMINISTRATION OF ANTERIOR HYPOPHYSEAL HORMONES AND GONADOTROPHIN PREPARATIONS TO HYPOPHYSECTOMIZED RATS

A crude human hypophyseal extract (HE), as well as human growth hormone (GH), ovine prolactin (PRL) and commercial preparations of ACTH, TSH, pregnant mare's serum gonadotrophins (PMS) and chorionic gonadotrophin (CG) were tested for their ability to induce the activities of cytoplasmic 17 beta-hydroxysteroid dehydrogenase and microsomal delta 4-5alpha-hydrogenase and to repress the activities of microsomal 3alpha- and 3beta-hydroxysteroid dehydrogenases in the liver of hypophysectomized rats. The activity of 17beta-hydroxysteroid dehydrogenase was not affected by any of the administered hormones. For the other enzymes, only PRL was effective in causing changes in the activities; the repressive effect on 3alpha-hydroxysteroid dehydrogenase activity was highly significant (P less than 0.001). These results indicate that PRL is involved in the regulation of at least some of the enzyme activities of hepatic steroid hormone metabolism.

The effect of different preparations of human growth hormone on plasma renin activity in normal males.

The effect of acute administration of 2 preparations of growth hormone (hGH) on plasma renin activity (PRA) was studied in normal volunteers. 4 IU of standard, commercially available hGH II, Kabi, Sweden) were injected im into each of four normal subjects, and the PRA was determined in the basal state and at 30, 60, 120, 180, and 240 min after injection. Free fatty acid (FFA) was determined basally and at 15 and 210 min post-injection. The study was repeated on a different day in the same group using highly purified hGH (hGH I, 4 IU) virtually free of arginine-vasopressin. Four other normal subjects received 12 IU standard hGH (hGH II, Kabi, Sweden). There was no significant difference in PRA values with 4 IU of either preparation or with 12 IU of hGH II in any of the groups, although mean PRA was elevated in two of the patients receiving 12 IU hGH II. A rise in FFA occurred in all subjects, indicating the biological activity of hGH preparations. The possible significance of these findings to the renin-angiotensin system found in acromegaly is considered.