kDa Growth Hormone Research Articles

Acute Endocrine Responses With Long-Term Weight Lifting in a 51-Year-Old Male Weightlifter.

Eserhaut, DA, Fry, AC, Stone, MH, and Kraemer, WJ. Acute endocrine responses with long-term weightlifting in a 51 year old male weightlifter. J Strength Cond Res XX(X): 000-000, 2024-To study the effects of long-term (>35 years) competitive weightlifting on acute endocrine activity, a 51-year-old male two-time Olympian in weightlifting (Subject A) was compared with highly trained young male weightlifters (controls, n = 23; age = 17.7 ± 0.3 years). Between 1400 and 1700 hours, subjects performed 15 maximum effort vertical jumps, a series of single repetitions of the snatch progressing to a maximal effort, and 3 × 10 snatch pulls at 60% 1 repetition maximum. Blood sampling occurred at 0700 hours, pre-exercise, and +5 and +15 minutes postexercise. Exercise performances were comparable between Subject A and controls and elicited similar lactate responses at +5 minutes (mmol·L-1; A = 9.2; controls = 8.1 ± 0.4). Resting testosterone at 0700 hours was lower for Subject A than for the controls (nmol·L-1; A = 13.9; controls = 25.4 ± 2.0), although acute responses at +5 minutes were comparable (A = 21.6; controls 18.3 ± 1.5). Cortisol responses (nmol·L-1; A = 468.4, controls = 540.6 ± 32.3) and testosterone:cortisol ratio (A = 0.0461; controls = 0.0376 ± 0.004) were similar at +5 minutes. Subject A showed no 22 kDa growth hormone response at +5 minutes, while controls exhibited a substantial increase (µg·L-1; A = 0.4; controls = 16.7 ± 2.6). β-endorphin responses were no different at +15 minutes for Subject A relative to controls (pmol·L-1; A = 30.1; controls = 33.8 ± 3.7). Resting and exercise-induced endocrine physiology is partially modified with aging despite long-term participation in competitive weightlifting. Importantly, Subject A's testosterone response was largely preserved.

A combined top-down and bottom-up LC-HRMS/MS method for the quantification of human growth hormone in plasma and serum

ObjectiveThe precise and accurate quantification of human growth hormone (GH) in plasma/ serum is crucial for the diagnosis and treatment of diseases like GH deficiency or acromegaly. However, the ligand-binding assays (LBAs) currently used for routine testing show considerable methodological variability. Here, we present a complementary, combined top-down and bottom-up LC-MS-based method to quantify (intact) GH in plasma and serum, which concurrently provides a basis for a MS-based analysis of GH in doping controls. DesignExtraction of GH from plasma/ serum was accomplished by protein precipitation, followed by an immunocapture step using protein A-coupled magnetic beads and a polyclonal anti-GH antibody. The intact protein was subsequently analyzed top-down on a 2D-LC-HRMS/MS system. In addition, sample extracts were digested with trypsin and analyzed for signal peptides corresponding to ‘total’, 22 kDa and 20 kDa GH (bottom-up). Both assays were validated according to current guidelines and compared to the GH isoform differential immunoassay used in routine doping control analysis. GH concentrations in serum samples of healthy adults, patients with acromegaly, and in samples obtained after administration of recombinant GH were analyzed as proof-of-principle. ResultsThe intact monomeric 22 kDa isoform of GH was selectively quantified in a representative working range of 0.5 to 10 ng/ml by top-down LC-HRMS/MS. Subsequent bottom-up analysis provided additional data on ‘total’ and 20 kDa GH. Top-down and bottom-up assay results for the 22 kDa isoform correlated well with the corresponding immunoassay results (R2 > 0.95). For a possible application of the method in an anti-doping context, the ratio between 22 kDa and ‘total’ GH was evaluated, indicating differences between the various donor groups, but only with limited significance. ConclusionThe top-down and bottom-up LC-HRMS/MS method developed here presents a valuable tool for the quantification of GH in plasma/ serum complementary to established LBAs used at present in clinical measurements. Albeit the examination of the GH isoform proportions by the LC-MS method does not yet allow for the assessment of GH abuse, the obtained findings provide an important basis to enable LC-MS-based GH analysis of doping control samples in the future.

GH1 C.291 + 34G>C in a Growth Hormone Deficient Pedigree

Background: Heterozygous loss of function mutations in GH1 are a cause of autosomal dominant isolated growth hormone deficiency (GHD) or IGHD type II. However, this condition is rare and a genotype/phenotype association is often based on single case or pedigree reports. Clinical Case: The male proband was born at 38 weeks following an uncomplicated pregnancy, birthweight 2585 gm (˗1.3SD), length 43 cm (˗2.7SD). At 20 months old: length ˗3.2SD, weight/length 16%. Peak growth hormone (GH) levels 4.42 ng/mL and 3.2 ng/mL following clonidine and arginine stimulation, respectively; cortrosyn-stimulated cortisol was 41.4 mcg/dL. Prolactin was 61.2 ng/mL (reference range 2.6 to 13.1 ng/mL); T4 and TSH levels were normal. Magnetic resonance imaging showed a flattened anterior pituitary gland, consistent with empty sella, with normal bright spot. He has had an intermittent and variable response to GH treatment due to noncompliance; recent recorded height was ˗3.6SD at 13.9 and he was early pubertal. Father had a history of childhood growth hormone treatment and is 151 cm tall, 2 paternal aunts did not receive treatment and are 145 cm tall, 3 children of these aunts are diagnosed with GHD. There is no maternal family history of GHD; mother is 155 cm. Proband testing revealed heterozygous GH1 c.291 + 34G>C (EGL Genetics), classified as a variant of unknown significance; father was also found to have this variant, mother was negative. Genetic testing for of additional family members is under consideration. Conclusion: Nine of 96 heterozygous GH1 variants are located in intron 3. Two have been classified as pathogenic and are predicted to cause exon skipping. Our proband’s variant cannot be considered a canonical splice site or in a mutational hot spot but has extremely low frequency in population databases. Functional studies clan clarify if our proband’s variant results in a 17.5 kDa GH isoform and dominant negative effect on full length GH production with potential detrimental effects on other anterior pituitary hormones. We present the second GHD pedigree with GH1 c.291 + 34G>C mutation. As in the previous report (1), no other hormone deficiencies are identified, despite the appearance of an empty sella for our proband. Reference: (1) Cho SY, Ki, CS, Park, HD, et al. Genetic investigation of patients with undetectable peaks of growth hormone after two provocation tests. Clin Endocrinol 2013; 78: 317-20.

SUN-LB62 Differential Regulation of 22 kDa and 20 kDa Growth Hormone Isoforms in Response to Acute Glucose Load and Moderate Intensity Exercise

Background: Among growth hormone (GH) isoforms, the 22kDa (22kD) is the most abundant (~90%), followed by 20 kDa GH ((20kD) ~10%). 22kD has known diabetogenic potential by increasing insulin resistance. Although of equal somatotropic activity, 20kD has been suggested to be less diabetogenic. Under physiological conditions, both isoforms are largely secreted in parallel, but data on regulation of both isoforms in the context of glucose load and utilization are scarce. Methods: We measured 20- and 22kD GH in fasted healthy adult females during two scenarios: (A) Premenopausal females (n = 25; age (mean (range)) 27.8 (22-52), body mass index (BMI) (kg/m2) 21.8 (18.2-27.3), insulin sensitivity index (ISI) 7.9 (3.2-19.7) underwent an oral glucose tolerance test (OGTT, 75g, sampling at baseline, 30, 60, 90, 120 and 180 minutes (min); (B) Premenopausal females within 1 year postpartum (n=28, age 36 (29-44), BMI 23.7 (19.4-41.3), ISI (7.2- (2.4-13.2)) performed moderate intensity exercise (fixed workload 60% VO2max, sampling at -15, -10, -5, 0, 3, 6, 10, 15, 20, 30 and 45 min and after 5, 10 and 15 min of resting). 20- and 22kD were measured by specific immunoassays (22kD: IDS-iSYS GH CLIA, limit of quantification (LoQ) 0.05 ng/mL; 20kD: in-house immunofluorometric assay (IFMA), LoQ 0.025 ng/mL), and the 20/22kD ratio was calculated. Results: As expected, both, 22kD and 20kD concentrations decreased after glucose load. However, the 20/22kD ratio increased significantly (Friedman test, P = 0.04, Kendall’s W 0.69, Conover’s post test: 0 vs. 60, 90 and 120 min; P = 0.018, 0.032 and 0.018, respectively. After 120 min, 20/22kD returned to baseline.With exercise, both, 20- and 22kD increased. In contrast to glucose load, the 20/22kD ratio significantly decreased (Friedman test, P = 0.025, Kendall’s W 0.59, Conover’s post test: -10 vs. 10 and 20 min; P = 0.015 and 0.022, respectively), reaching a nadir at 10 min and returning to the baseline after cessation of exercise (20 vs. 50, 55 and 60 min; P = 0.02, 0.005 and 0.016, respectively. Conclusion: Glucose load and exercise lead to opposite changes in the 20/22kD ratio, suggesting a tight regulation by glucose demands. We speculate that the putative less diabetogenic isoform (20kD) is preferentially secreted during situations of glucose excess, while the opposite happens during exercise, when increased energy demands are associated with predominant upregulation of the more diabetogenic 22kD GH.

Laboratory investigations in the diagnosis and follow-up of GH-related disorders.

In addition to auxiological, clinical and metabolic features measurements of growth hormone (GH) and insulin-like growth factor I (IGF-I) complement our tools in diagnosis and follow-up of GH-related disorders. While comparably robust during the pre-analytical phase, measurement and interpretation of concentrations of both hormones can be challenging due to analytical issues and biological confounders. Assay methods differ in terms of antibody specificity, interference from binding proteins, reference preparations and sensitivity. GH assays have different specificity towards different GH-isoforms (e.g. 20 kDa GH, placental GH) and interference from the GH antagonist Pegvisomant. The efficacy to prevent binding protein interference is most important in IGF-I assays. Methodological differences between assays require that reference intervals and diagnostic cut-offs are assay-specific. Among biological variables, pubertal development and age are most relevant for IGF-I, making detailed reference intervals mandatory for interpretation. GH has pulsatile secretion and short half-life. Its concentration is modified by acute factors such as stress, exercise and sleep, but also by intake of oral estrogens and anthropometric factors (e.g. BMI). Other GH dependent biomarkers such as free IGF-I, IGF binding protein 3 (IGFBP 3) and acid labile subunit (ALS) have been proposed. Their concentrations largely mirror the information obtained through measurement of IGF-I, but their measurement can be helpful in particular situations. In this review, we describe the evolution of analytical methods to measure biomarkers of GH action, the impact of the methodological changes on laboratory results and the need to include biological variables in their interpretation. Arch Endocrinol Metab. 2019;63(6):618-29.

Detection and differentiation of 22 kDa and 20 kDa Growth Hormone proteoforms in human plasma by LC-MS/MS

Human growth hormone (GH) is suspected to be widely and illegally used in sport to improve athletes' performance. For the detection of GH abuse, blood samples are screened for abnormal ratios between the 22 and 20kDa GH proteoforms that demonstrate the administration of the synthetic hormone. Current detection methods are based on classical immunoassays as they provide sufficient sensitivity for the detection of GH proteoforms. These antibody based methods, however, suffer from unclear selectivity and potential cross-reactivity towards similar proteins. For unambiguous GH detection, we report a Mass Spectrometry ImmunoAssay (MSIA) that first enriches GH from plasma with an antibody of relatively low specificity, and subsequently quantifies the 22 and 20kDa proteoforms by Selected Reaction Monitoring (SRM) LC-MS/MS analysis. This method proved superior to an antibody-free strategy based on GH purification by protein precipitation. Using GH-MSIA we successfully quantified the 22/20kDa GH ratio in post-exercise capillary plasma extracted from two individuals. This GH-MSIA is applicable to anti-doping and GH-related disease analysis.

Non-classical Signalling of Growth Hormone in the Chick Neural Retina?

Growth hormone (GH) signalling is traditionally via a ‘classical’ GH-receptor (GHR) that is present in the plasma membrane of target cells. GHRs are present in the neural retina of early chick embryos (within the first trimester of incubation) but these are unlikely to be endocrine target sites of GH action, as pituitary GH is not secreted into the bloodstream until late in development (approximately day 17 of the 21 days incubation period) and because the blood–ocular barrier would block the entry of systemic GH into the retina. As GH is expressed in the early neural retina, retinal GHRs are thus likely to be autocrine or paracrine sites of GH action. The most abundant GH moieties in the neural retina are, however, unlikely to activate ‘classical’ GHRs in the neural retina, as they are submonomeric molecules (of 15 kDa and 16.5 kDa) that fail to bind to plasma membrane receptor (15 kDa GH) or lack the stoichiometric requirements likely required for GHR binding (16.5 kDa GH). Nevertheless, 15 kDa GH is biologically active in chicks and the immunoneutralisation of endogenous 16.5 kDa GH induces biological activity. It is therefore possible that GH in the neural retina of early chick embryos acts non-classically or via ‘non-classical’ GHRs. These possibilities are the focus of this brief review.

Long-Acting Growth Hormone

Human growth hormone (GH) is a 22 kDa protein synthesized and secreted in the anterior pituitary gland. GH synthesis and release is induced by hypothalamic GHreleasing hormone (GHRH) and ghrelin and attenuated by somatostatin and negative feedback by insulin-like growth factor (IGF)-I [1]. The secretion of GH is episodic with several secretory peaks. The majority of spontaneous peaks occur during sleep, but it is also influenced by age, gender, intercurrent and chronic illnesses, and by nutritional status [1, 2]. GH is required for linear growth during childhood and adolescence. GH regulates the production of IGF-I, which is mostly produced in the liver, and, together, GH and IGFI stimulate skeletal growth [1]. GH also has profound metabolic effects throughout life [2]. Through its anabolic, lipolytic, and antinatriuretic effects, it increases muscle mass and bone formation, reduces fat mass, and increases total body water. GH augments metabolic activity, resulting in a rapid and large increase in resting energy expenditure and fat oxidation, and protein synthesis increases [3]. Additionally, the peripheral conversions of thyroxine (T4) to triiodothyronine (T3) and of cortisol to the inactive cortisone rise. Furthermore, GH increases insulin resistance and causes hyperinsulinaemia. The lipolytic effect, and hence the impact upon body composition, is of course most readily appreciated during the initial years of replacement therapy, since a new steady state will be reached [4]. The diagnosis of GH deficiency (GHD) requires demonstration of a blunted response to stimulation tests according to specific diagnostic criteria [5, 6]. Insulininduced hypoglycaemia (insulin tolerance test [ITT]) is the reference golden standard for diagnosing GHD, but the GHRH–arginine test and glucagon test are commonly used, with different cut-offs applied for the different tests. IGF-I is correlated with GH secretion, but because of overlap in IGF-I levels in healthy individuals and patients with GHD, IGF-I is not useful for diagnosing GHD [7]. However, IGFI expressed as ageand gender-adjusted standard deviation score (IGF-I-SDS) is routinely used for monitoring GH dosing. GHD during childhood results in growth retardation, as well as an abnormal body composition, with more body fat than lean body mass, and decreased physical capacity and quality of life. Treatment with GH in children with GHD has been well established for some 20–30 years and has, in several studies, been verified to promote linear growth and improve metabolism [6]. In fact, replacement of GH today extends long into adult life and even into senescence. Nowadays, indications include GHD in both children and adults, children with Prader–Willi Syndrome, Turner Syndrome, and Noonan Syndrome, as well as idiopathic short stature (ISS), children born small for gestational age (SGA), and children with chronic renal insufficiency (CRI). Treatment with GH became possible in the late 1950s following the isolation and purification of GH from human cadaver pituitary glands by Raben [8]. Since only extracts from the human pituitary gland were effective in humans, the availability of GH for clinical use was very limited. Following the introduction of the biosynthetically derived natural sequence (22 kDa GH) in the mid 1980s, there has been no shortage of GH for clinical use. C. Hoybye Department of Endocrinology, Metabolism and Diabetology, Karolinska University Hospital, Stockholm, Sweden

High sensitivity mass spectrometric quantification of serum growth hormone by amphiphilic peptide conjugation

Amphiphilic peptide conjugation affords a significant increase in sensitivity with protein quantification by electrospray-ionization mass spectrometry. This has been demonstrated for human growth hormone (GH) in serum using N-(3-iodopropyl)-N,N,N-dimethyloctylammonium iodide as derivatizing reagent. The signal enhancement achieved is up to a factor of 5-6 and enables extension of the applicable concentration range down to the very low concentrations (≤ 1.0 µg/L) as encountered with clinical glucose suppression tests for patients with acromegaly. The method has been validated using a set of serum samples spiked with known amounts of recombinant 22 kDa GH in the range of 0.48 to 7.65 µg/L. The coefficient of variation (CV) calculated based on the deviation of results from the expected concentrations was 3.5%. The limit of detection (LoD) was determined as 0.1 µg/L and the limit of quantification (LoQ) as 0.4 µg/L. The potential of the method as a tool in clinical practice has been demonstrated with patient samples of about 1 µg/L.

OR07-2 Simultaneous stimulation of human 20 kDa growth hormone and 22 kDa growth hormone secretion by ghrelin

The World Anti Doping Agency has banned the use of recombinant human growth hormone (rhGH). Regardless, rogue athletes may use rhGH in pursuit of improved performance. Current immunoassaybased detection of illicit use of rhGH is complicated by the short half-life of injected rhGH, by the chemical identity of rhGH and a native growth hormone (GH), and by the physiologically high peaks of GH that occur with the pulsatile secretion of GH by a normal pituitary. We hypothesized that injection of rhGH changes the expression patterns of GHmessenger RNA (mRNA) in circulating white blood cells and of microRNA (miRNA) in plasma. Objective: To measure circulating GH mRNA and miRNAs in humans. Methods: We used reverse-transcription quantitative polymerase chain reaction (RT-qPCR) with nested primers to detect and quantify GHmRNA.We usedmiRNAmicroarrays to identify rhGH-dependent miRNA expression and then measured the concentrations of selected miRNAs by RT-qPCR. Following Institutional Review Board approval, we obtained whole-blood or plasma, from 9 healthy volunteers and from 36 patient volunteers. All patients had known or suspected disorders of growth hormone production including panhypopituitarism treated with physiologic doses of rhGH (0.2 to 0.8mg/day) and acromegaly. Results: GH mRNA was quantified in blood from all healthy volunteers and patients with disorders of GH at concentrations ranging from 36 to 4815 copies/100 mL blood. The intra-individual GH mRNA concentration varied less than 3-fold in 17 samples collected over the period of one year in a healthy, athletic volunteer; however, variability increased markedly in the group with pituitary disorders. Initially, 9 specimens from: 3 patients receiving therapeutic growth hormone replacement, 4 patients with acromegaly, and 2 normal controls, were subject to plasma-derived miRNA microarrays. Of 1105 miRNA’s measured in each microarray, 3 were present in plasma of all rhGH replacement users and undetectable in all patients with acromegaly and normal controls. Conclusions: The narrow intra-individual range of its concentration suggests that GH mRNA in blood is not present as a result of pulsatile secretion and thus is worthy of study as a marker of doping with supraphysiologic amounts of rhGH. Further investigation of miRNA in plasma should be performed on samples obtained before and after administration of supraphysiologic amounts of rhGH to confirm the preliminary results in patients receiving therapeutic replacement doses of rhGH.

Ghrelin Stimulation of Growth Hormone Isoforms: Parallel Secretion of Total and 20-kDa Growth Hormone and Relation to Insulin Sensitivity in Healthy Humans

The 20-kDa human GH (hGH) is produced in the pituitary by alternative splicing of the hGH-N gene. The 20-kDa hGH promotes growth similarly to 22-kDa or total hGH, the predominant form in circulation, but the relative effects of these isoforms on glucose metabolism have been debated. To investigate the effect of ghrelin on 20-kDa and total hGH secretion in healthy, nonobese subjects. We also studied associations between basal GH concentration and fasting glucose and insulin as well as between dynamic GH secretion and insulin sensitivity. Synthetic human acyl ghrelin (0.2 or 0.6 nmol/kg · h) or saline was infused in random order in 14 healthy subjects (six males, eight females; age 27.7 ± 6.3 yr; body mass index 22.0 ± 2.7 kg/m(2), mean ± SEM) on 3 separate days. Ghrelin was infused for 45 min to achieve steady-state levels and continued through a 3-h frequently sampled i.v. glucose tolerance test. Insulin sensitivity index was quantified using the minimal model of glucose kinetics. Basal 20-kDa and total GH concentrations were 0.4 ± 0.1 and 2.2 ± 0.4 ng/ml, respectively, with a 20-kDa to total GH ratio of 0.13 ± 0.02. Females had significantly higher baseline GH levels. Ghrelin administration increased 20-kDa and total GH levels in a parallel and dose-dependent fashion, with no significant change in the ratio of the isoforms. Basal 20-kDa and total GH levels were negatively correlated with fasting glucose, insulin, and homeostasis model assessment of insulin resistance. During the frequently sampled iv glucose tolerance test, GH secretion was positively correlated with insulin sensitivity index with saline infusion. Ghrelin dose-dependently increases endogenous 20-kDa and total GH secretion in a parallel fashion in healthy subjects. Both basal and stimulated levels of the different GH isoforms were positively associated with insulin sensitivity in this cohort of healthy men and women.

Growth Hormone in Sports: Detecting the Doped or Duped

Background: Doping with growth hormone (GH) is banned; however, there is anecdotal evidence that it is widely abused. GH is reportedly often used in combination with anabolic steroids at high doses for several months. Development of a robust test for detecting GH has been challenging since recombinant human 22-kDa GH used in doping is indistinguishable analytically from endogenous GH and there are wide physiological fluctuations in circulating GH concentrations. Discussion: One approach to GH testing is based on measurement of different circulating GH isoforms using immunoassays that differentiate between 22-kDa and other GH isoforms. Administration of 22-kDa GH results in a change in its abundance relative to other endogenous pituitary GH isoforms. The differential isoform method is, however, limited by its short time window of detection. A second approach that extends the time window of detection is based on detection of increased levels of circulating GH-responsive proteins, such as the insulin-like growth factor (IGF) axis and collagen peptides. As age and gender are the major determinants of variability for IGF-I and the collagen markers, a test based on these markers must take these factors into account. Extensive data now validate the GH-responsive marker approach, and implementation is largely dependent on establishing an assured supply of standardized assays. Conclusions: Robust tests are available to detect GH and enforce the ban on its abuse in sports. Novel approaches that include gene expression and proteomic profiling must continue to be pursued to expand the repertoire of testing approaches available and to maintain deterrence of GH doping.

SECRETION AND PROLIFERATIVE EFFECTS OF CHICKEN GROWTH HORMONE IN TESTICULAR CELL CULTURES TREATED WITH GHRH

Event Abstract Back to Event SECRETION AND PROLIFERATIVE EFFECTS OF CHICKEN GROWTH HORMONE IN TESTICULAR CELL CULTURES TREATED WITH GHRH Carlos G. Martínez-Moreno1, Luz M. López-Marín2, Martha E. Carranza1, Carlos Arámburo1 and Maricela Luna1* 1 Universidad Nacional Autonoma de Mexico, Institute of Neurobiology, Dept. of Molecular and Cellular Neurobiology, Mexico 2 Universidad Nacional Autónoma de México, Centro de Física Aplicada y Tecnología Avanzada, Mexico Recent findings showed the cytoplasmic and nuclear distribution of growth hormone (GH) and its receptor (GHR) in chicken testicular germ cells, suggesting an intracrine, autocrine and/or paracrine role for this hormone during spermatogenesis. The expression of testicular GH may be modulated by a variety of factors, including growth hormone releasing hormone (GHRH) and Pit-1. We analyzed the effect of GHRH (1, 10 and 100 nM, for 2 h) on GH release into the media of primary cell cultures derived from chicken testes at two developmental stages: during puberty (20 weeks, PC) and sexual maturity (32 weeks, MC). Released GH increased after GHRH treatment both in PC media (10.8±1, 13.4±2 and 14.5±2 ng/ml) and MC media (5.83±2.05, 7.6±0.6 and 8.5±2 ng/ml) in comparison to the corresponding controls (7.4±1 and 3.2±0.7 ng/ml, respectively). Western blot analyses were performed to characterize whether GHRH modulation involved secretion of particular GH isoforms. Interestingly, 15 kDa and 26 kDa GH variants were differentially modulated after 100 nM GHRH treatment, resulting in a 2.5 fold-increase in 15 kDa GH secretion, whereas a significant decrease was found for the 26 kDa variant release. On the other hand, GHRH (1, 10, 100 nM) stimulated proliferation of testicular cells in culture (2.7-, 3.6- and 2.0-fold, respectively, in comparison to the controls) as determined by the PCNA assay. Using the 3H-thymidine incorporation assay GH also increased the proliferation of testicular cells (by 118.7±18.1%, and 170.8±28%, and 102.1±15.7% at 1, 10 and 100 nM, respectively, when compared to 96.1±12.4% in the untreated controls). Altogether, these results suggest a role of GHRH and GH during chicken testicular cell proliferation, probably through autocrine/paracrine mechanisms. Acknowledgements This work was supported by grants CONACyT (F1-60296) and PAPIIT-UNAM (IN-210209), graduate fellowships from CONACyT (185024) and PDCB-UNAM and Alejandro Bayón Fund established by CA Keywords: autocrine/paracrine mechanisms, extrapituitary growth hormone, GHRH, Growth Hormone, proliferation, testicular cell cultures, testicular growth hormone variants Conference: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology, Ann Arbor, United States, 13 Jul - 16 Jul, 2011. Presentation Type: Poster Topic: Gonadal development and gamete maturation Citation: Martínez-Moreno CG, López-Marín LM, Carranza ME, Arámburo C and Luna M (2011). SECRETION AND PROLIFERATIVE EFFECTS OF CHICKEN GROWTH HORMONE IN TESTICULAR CELL CULTURES TREATED WITH GHRH. Front. Endocrinol. Conference Abstract: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology. doi: 10.3389/conf.fendo.2011.04.00094 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 23 Jul 2011; Published Online: 09 Aug 2011. * Correspondence: Dr. Maricela Luna, Universidad Nacional Autonoma de Mexico, Institute of Neurobiology, Dept. of Molecular and Cellular Neurobiology, Queretaro, Queretaro, 76230, Mexico, lunam@servidor.unam.mx Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract Supplemental Data The Authors in Frontiers Carlos G Martínez-Moreno Luz M López-Marín Martha E Carranza Carlos Arámburo Maricela Luna Google Carlos G Martínez-Moreno Luz M López-Marín Martha E Carranza Carlos Arámburo Maricela Luna Google Scholar Carlos G Martínez-Moreno Luz M López-Marín Martha E Carranza Carlos Arámburo Maricela Luna PubMed Carlos G Martínez-Moreno Luz M López-Marín Martha E Carranza Carlos Arámburo Maricela Luna Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. 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Influence of intravenous administration of growth hormone releasing peptide‐2 (GHRP‐2) on detection of growth hormone doping: growth hormone isoform profiles in Japanese male subjects

Administration of exogenous 22 kDa recombinant human growth hormone (rhGH) suppresses the non-22 kDa pituitary growth hormone (GH) secretion by negative feedback; then, the elevated 22 kDa GH to non-22 kDa GH ratio (Rec/Pit ratio) can be utilized to detect doping with rhGH (isoform differential immunoassay). The influence of intravenous administration of growth hormone releasing peptide GHRP-2 on the isoform differential immunoassay for detecting rhGH doping has been investigated.In this study, a reference population (n=100) was used, with 0.04 mg/kg rhGH subcutaneous administration (n=5), 100 μg of GHRP-2 intravenous administration (n=10) and 0.04 mg/kg rhGH combined with 100 μg of GHRP-2 (n=10) in Japanese male subjects. The results indicated that the low dose (0.04 mg/kg) of rhGH led to significantly increased Rec/Pit ratio compared with the Japanese reference limit (P < 0.001). Because GHRP-2 dose led to increases in concentrations of both recombinant GH (recGH) and pituitary GH (pit GH), no significant change in the Rec/Pit ratio was observed (P > 0.05). In a combined administration study, after GHRP-2 dose the Rec/Pit ratios decreased to 39.9-43.9% compared with the elevated ratio caused by the rhGH dose.The results indicated that GHRP-2 administration cannot only be detected by the isoform differential immunoassay but also masks rhGH doping. The analysis of GHRP-2 was found to be suitable for compensating for the disadvantages of the isoform differential immunoassay because GHRP-2 and its metabolite (AA-3) in urine could be detected during the periods of masking of the Rec/Pit ratio by means of liquid chromatography/tandem mass spectrometry.

Growth Hormone (GH)-Releasing Hormone Increases the Expression of the Dominant-Negative GH Isoform in Cases of Isolated GH Deficiency due to GH Splice-Site Mutations

An autosomal dominant form of isolated GH deficiency (IGHD II) can result from heterozygous splice site mutations that weaken recognition of exon 3 leading to aberrant splicing of GH-1 transcripts and production of a dominant-negative 17.5-kDa GH isoform. Previous studies suggested that the extent of missplicing varies with different mutations and the level of GH expression and/or secretion. To study this, wt-hGH and/or different hGH-splice site mutants (GH-IVS+2, GH-IVS+6, GH-ISE+28) were transfected in rat pituitary cells expressing human GHRH receptor (GC-GHRHR). Upon GHRH stimulation, GC-GHRHR cells coexpressing wt-hGH and each of the mutants displayed reduced hGH secretion and intracellular GH content when compared with cells expressing only wt-hGH, confirming the dominant-negative effect of 17.5-kDa isoform on the secretion of 22-kDa GH. Furthermore, increased amount of 17.5-kDa isoform produced after GHRH stimulation in cells expressing GH-splice site mutants reduced production of endogenous rat GH, which was not observed after GHRH-induced increase in wt-hGH. In conclusion, our results support the hypothesis that after GHRH stimulation, the severity of IGHD II depends on the position of splice site mutation leading to the production of increasing amounts of 17.5-kDa protein, which reduces the storage and secretion of wt-GH in the most severely affected cases. Due to the absence of GH and IGF-I-negative feedback in IGHD II, a chronic up-regulation of GHRH would lead to an increased stimulatory drive to somatotrophs to produce more 17.5-kDa GH from the severest mutant alleles, thereby accelerating autodestruction of somatotrophs in a vicious cycle.

Quantification of growth hormone in serum by isotope dilution mass spectrometry

Interassay variation of antibody-based routine tests hampers comparability of measurement results for growth hormone (GH) between different laboratories and decision making in clinical practice. Here it is demonstrated that quantification of GH by isotope dilution mass spectrometry (IDMS) constitutes a way to obtain precise and reliable results that can be referred to in evaluation of performance of commercial test kits. With the IDMS method developed, tryptic cleavage products YSFLQNPQTSLCFSESIPTPSNR (T6) and LEDGSPR (T12) of GH are quantified by liquid chromatography tandem mass spectrometry (LC–MS/MS) using isotopically labeled forms of the peptides as internal standards. The GH cleavage fragments are obtained by whole serum tryptic proteolysis and then extracted from the resulting mixture by semipreparative reversed-phase LC followed by strong cation exchange chromatography. Analysis of blank serum spiked with recombinant 22-kDa GH at different concentration levels would result in a mean recovery of 101.6%, a standard deviation (SD) of 2.5%, a combined uncertainty (uc) of 3.0%, and a limit of quantification (LOQ) of 1.7μg/L when quantifying T6 as a GH-derived fragment, whereas recovery=100.7%, SD=2.4%, uc=2.5%, and LOQ=2.7μg/L were found with T12. The potential to acquisition of reference values is exemplified by application to serum materials used in a recent quality assessment exercise for routine laboratories.

Quantification of growth hormone in serum by isotope dilution mass spectrometry

AbstractInter-assay variation of antibody based routine tests is hampering comparability of measurement results for growth hormone (GH) between different laboratories and decision making in clinical practice. Here it is demonstrated, that quantification of GH by isotope dilution mass spectrometry (IDMS) constitutes a way to precise and reliable results which can be referred to in evaluation of performance of commercial test kits. With the IDMS method developed, tryptic cleavage products YSFLQNPQTSLCFSESIPTPSNR (T6) and LEDGSPR (T12) of GH are quantified by LC/MS-MS using the isotopically labeled forms of the peptides as internal standards. The GH cleavage fragments are obtained by whole-serum tryptic proteolysis and then extracted from the resulting mixture by semi-preparative reversed phase liquid chromatography followed by strong cation-exchange chromatography. Method validation basing on recovery of recombinant 22 kDa GH spiked to blank serum in defined amounts covering the intended concentration range (3-30 µg/L) would yield mean recoveries of 101.6% (100.7%), standard deviations of 2.5% (2.4%) and combined uncertainties (u~c~) of 3.0% (2.5%) if quantifying T6 (T12) as GH derived fragments, while the LOQ were 1.7 µg/L (2.7 µg/L). Potential to acquisition of reference values is exemplified by application to serum materials used in a recent quality assessment exercise for routine laboratories.

Identification of Human Pituitary Growth Hormone Variants by Mass Spectrometry

The heterogeneity of human endogenous growth hormone (GH) is used in doping control analysis to distinguish it from the homogeneous recombinant analogue in plasma samples. Pituitary GH variants were characterized by gel electrophoresis and mass spectrometry. Besides 22 and 20 kDa isoforms, fragments of 9 and 12 kDa were identified and a glycosylated 23 kDa GH variant was elucidated to bear a HexHexNac 2 NeuAc modification presumably located at Thr 60.

A robust test for growth hormone doping – present status and future prospects

Although doping with growth hormone (GH) is banned, there is anecdotal evidence that it is widely abused. GH is reportedly used often in combination with anabolic steroids at high doses for several months. Development of a robust test for GH has been challenging because recombinant human 22 kDa (22K) GH used in doping is indistinguishable analytically from endogenous GH and there are wide physiological fluctuations in circulating GH concentrations. One approach to GH testing is based on measurement of different circulating GH isoforms using immunoassays that differentiate between 22K and other GH isoforms. Administration of 22K GH results in a change in its abundance relative to other endogenous pituitary GH isoforms. The differential isoform method has been implemented; however, its utility is limited because of the short window of opportunity of detection. The second approach, which will extend the window of opportunity of detection, is based on the detection of increased levels of circulating GH-responsive proteins, such as insulin-like growth factor (IGF) axis and collagen peptides. Age and gender are the major determinants of variability for IGF-I and the collagen markers; therefore, a test based on these markers must take age into account for men and women. Extensive data is now available that validates the GH-responsive marker approach and implementation is now largely dependent on establishing an assured supply of standardized assays. Future directions will include more widespread implementation of both approaches by the World Anti-Doping Agency, possible use of other platforms for measurement and an athlete's passport to establish individual reference levels for biological parameters such as GH-responsive markers. Novel approaches include gene expression and proteomic profiling.

Characterisation of the 5 kDa growth hormone isoform

Objectives: The 5 kDa N-terminal fragment of 43 amino acids of human growth hormone (GH) shows a specific and significant in-vivo insulin-like activity. This isoform can be easily obtained by solid phase synthesis methods. Our objective in this study is to describe this procedure in detail and to provide structural information of the protein.Methods: Solid phase synthesis was employed for the synthesis of the 5 kDa GH isoform. Circular dichroism and limited proteolysis have been carried out to provide structural information about the folded state of the protein in solution. Surface plasmon resonance was used to compare the structural equivalence between the synthetic protein and a proteolytic homologue at an antibody binding level. For this purpose, a murine monoclonal antibody specific for the 5 kDa isoform was generated and characterised employing this and several other GH isoforms.Results: Circular dichroism and proteolysis results suggested that the C-terminal segment of the 5 kDa protein folds in an α-helix. The comparison of the synthetic product to its proteolytic homologue at an antibody binding level suggested structural equivalency. A highly specific antibody against the 5 kDa GH isoform was generated with null cross-reactivity for 17, 20 and 22 kDa isoforms. Kinetic data on the interaction with the synthetic 5 kDa GH was obtained.Conclusions: The structure of the protein appears to be different in comparison to when it is included within the 22 kDa GH isoform. Finally, a highly specific antibody has been generated. The possible significance of the 5 kDa protein as a potential agent for obesity-related diseases is discussed.