Administration Of Recombinant Growth Hormone Research Articles

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.

Effect of systemic administration of recombinant growth hormone on rotator cuff repair a rat model study

Shoulder rotator cuff injuries are prevalent among the elderly. Rotator cuff tears, like osteoarthritis, are an inevitable aspect of aging. The only difference is that soft tissue rather than cartilage is involved in the degenerative process.We hypothesized that exogenous systemic administration of recombinant growth hormone would increase the concentration of various growth factors involved in rotator cuff healing,Forty-eight Sprague-Dawley rats (Charles River, male, 400-450 g) were subjected to unilateral detachment repair of the supraspinatus and infraspinatus tendons at Konkuk University per Institutional Animal Care Committee recommendations.The rats were divided in two groups one were injected with recombinant GH and controls. Initially an artificial rotator cuff tear was created and later repaired with vicryl 4-0. The animals were sacrificed at 1, 3, 6, and 12 weeks. The rotator cuff was removed enmasse. Both groups were compared histologically and immunologically for collagen 1 and 3. Statistical analysis between both the groups was done using SPSS software.The recombinant GH helped in early healing of rotator cuff tears if injected systemically. More clinical trials need to be done before the exact dose can be determined for humans.

Coupling Complete Blood Count and Steroidomics to Track Low Doses Administration of Recombinant Growth Hormone: An Anti-Doping Perspective.

Growth Hormone (GH) under its human recombinant homologue (rhGH), may be abused by athletes to take advantage of its well-known anabolic and lipolytic properties; hence it is prohibited in sports by the World Anti-Doping Agency. Due to the rapid turnover of rhGH, anti-doping screening tests have turned to monitor two endocrine biomarkers (IGF-I and P-III-NP), but unfortunately, they show population-wise variability, limiting the identification rate of rhGH users. Previous studies have evidenced the numerous effects of GH on human physiology, especially in hematopoiesis and steroidogenesis. In this work, aiming to discover novel physiological rhGH biomarkers, we analyzed the complete blood count and the steroidomics profile of healthy, physically active, young males treated either with EPO + rhGH or EPO + placebo. The time-trends of these two physiological routes have been analyzed through geometric trajectory analysis (GTA) and OPLS-DA. Individuals supplemented with micro-doses of rhGH exhibited different leukopoietic and steroidal profiles compared to the control population, suggesting a role of the rhGH in both pathways. In the article, hypotheses on the observed differences are discussed according to the most recent literature and compared to results in animal models. The use of leukopoietic and steroidal biomarkers together with endocrine biomarkers (IGF-1 and P-III-NP) allows to correctly classify over 98% of samples with no false positives, miss-classifying only one single sample (false negative) over a total of 56; a promising result, if compared to the current rhGH detection strategies.

Doping and GH

Abuse of growth hormone (GH) in sport is evidenced by customs and police, and more recently through testing in anti-doping laboratories. The popularity of GH among cheaters contrasts to the difficulty to prove performance-enhancing effects in controlled studies. Earlier results from replacement therapy in GH deficient adults indicated effects on muscle strength, which were not confirmed in meta-analyses. Even less evidence is available from healthy, trained adults. Obviously, it is impossible to mimic doses used by dopers in controlled studies. Changes in body composition induced by GH lipolytic actions could be helpful in some disciplines. Biochemical detection of doping with recombinant GH remained impossible for decades because of the short half-life in circulation and the similarity of pharmaceutical preparations to endogenous GH. The “marker method” uses changes in GH dependent biomarkers with longer half-lives, and some athletes have been banned because concentrations of IGF-I or P-III-P (a marker of bone and collagen turnover) were grossly elevated. High inter- and intra-individual variability of the biomarkers remains problematic. The “isoform-method” detects changes in molecular isoforms of GH occurring after administration of recombinant GH. This consists of the 22 kD isoform only, while the pituitary secretes a wide spectrum of GH isoforms. A respective change in circulating GH isoforms can be measured by specific assays. Window of detection is limited, but targeted out of competition testing continuously detects cheating athletes. Associated sanctions have been upheld in front of the court of arbitration is sports, and the method is widely implemented today.

Growth and survival of giant gourami juvenile immersed in freshwater containing growth hormone

<p>Growth of giant gourami is relatively slow. Administration of recombinant growth hormone (rGH) by several<br />times immersion in water containing 0.9% NaCl for one hour can significantly increase the growth. In this<br />study, giant gourami juvenile were immersed in freshwater containing 120 mg/L rGH to determine the long<br />immersion time that provides higher growth and survival. Juvenile at seventh-day-old after egg yolk absorbed<br />was immersed for 0.5, 1.0, 2.0, and 3.0 hours in rGH solution. As controls, fish was immersed in freshwater<br />without rGH (control 1: C1), and water containing 0.9% NaCl, and 120 mg/L rGH (control 2: C2). Fish was<br />kept under controlled conditions in 20 L aquarium for three weeks, then in 50 L aquarium until at end of<br />experiment, and fed at satiation. The results showed that highest growth in body weight, survival, and biomass<br />(p<0.05) were obtained in rGH immersion treatment for 0.5 hour. Biomass of fish immersed by rGH for 0.5<br />hour was about 32% higher compared to control C1, and 13% compared to C2. Survival of the 0.5 hour rGHimmersed<br />fish (100%) was similar to control C1 and C2. Thus, immersion of giant gourami juvenile in<br />freshwater containing rGH for 0.5 hour allowed highest growth rate, and this method is relatively easy to be<br />adopted by fish hatchery.</p><p>Keywords: immersion, hormone, growth, freshwater, giant gourami</p>

Effects of growth hormone and ultrasound on mandibular growth in rats: MicroCT and toxicity analyses

It has been shown by previous studies that mandibular growth can be enhanced by the systemic administration of recombinant growth hormone (rGH) and/or local application of therapeutic low intensity pulsed ultrasound (LIPUS). The purpose of this study was to determine if local injection of rGH and application of LIPUS to the temporomandibular joint (TMJ) would synergistically enhance mandibular growth. In an animal study, the effect of rGH, LIPUS, and combination of rGH and LIPUS on male Sprague–Dawley rats was observed. Mandibular growth was evaluated by measuring total hemimandibular and condylar bone volume and bone surface area as well as condylar bone mineral density (BMD) after 21 days on dissected rats’ mandibles using micro-computed tomography (MicroCT). The expression of c-jun mRNA extracted from the liver of each of these rats was also quantified by real-time polymerase chain reaction to evaluate possible systemic effect of local rGH administration. Significant growth stimulation was observed in the mandibular and condylar bone of the animals treated with rGH, LIPUS, and rGH/LIPUS combined when compared with the control group. Bone volume, surface area, condylar bone mineral density, and c-jun expression were also compared between the treatment groups and the control in the liver. The results suggest that mandibular growth may be enhanced by injection of rGH or LIPUS application. The current study although showed synergetic effect of rGH and LIPUS application in increasing mandibular condylar head length, there was no significant changes in mandibular bone volume using both treatments together when compared to the two individual treatments. Moreover, combined rGH and LIPUS decreased condylar bone mineral density than each treatment separately. Future research could be directed to investigate the effects of different rGH doses and/or different LIPUS exposures parameters on lower jaw growth.

The effects of two weeks of recombinant growth hormone administration on the response of IGF-I and N-terminal pro-peptide of collagen type III (P-III-NP) during a single bout of high resistance exercise in resistance trained young men

ObjectiveRecombinant human growth hormone (rhGH) is used by some athletes and body builders with the aim of enhancing performance, building muscle and improving physique. Detection of the misuse of rhGH has proved difficult for a number of reasons. One of these is the effect of preceding exercise. In this randomised, double blind placebo-controlled study, we determined the effects of rhGH administration in male amateur athletes on two candidate markers of rhGH abuse, IGF-I and N-terminal pro-peptide of collagen type III (P-III-NP), following a bout of weightlifting exercise. DesignSixteen men entered a four-week general weight training programme to homogenise their activity profile. They then undertook repeated bouts of standardised leg press weightlifting exercise (AHRET-acute heavy resistance exercise test). Blood samples were taken before and up to one hour after the AHRET. After the first laboratory visit (Test 1), the subjects were randomly assigned to receive daily injections of either rhGH (0.1IUkg−1day−1) or placebo for two weeks. The AHRET was repeated after the two-week dosing period (Test 2) and a further test was undertaken following a one-week washout (Test 3). ResultsThere was no effect of exercise on either IGF-I or P-III-NP in any test. Both markers were markedly elevated at Test 2 (p<0.001), with P-III-NP remaining elevated at Test 3 in the GH administration group (p<0.05). Application of the GH-2000 discriminant function positively identified GH administration in 17 of 40 blood samples taken at Test 2 from the rhGH group and none from the placebo group. ConclusionThe data show that rhGH results in elevated levels of IGF-I and P-III-NP in well-trained individuals and that leg press weightlifting exercise does not affect these markers. The GH-2000 discriminant function identified four of eight subjects taking rhGH with no false positive results.

726 Leri-Weill Dyschondrosteosis - A Case of Complete Deletion of One of the Copies of Shox Gene

IntroductionLeri-Weill dyschondrosteosis (LWD), is a dominantly inherited skeletal dysplasia with disproportionate short stature owing to mesomelic shortening of the forearm and lower leg and Madelung deformity of the arm is...

Growth, Chronic Kidney Disease and Pediatric Kidney Transplantation: Is It Useful to Use Recombinant Growth Hormone in Colombian Children With Renal Transplant?

Kidney transplantation has become the best treatment for children with chronic kidney disease (CKD). In recent times, knowledge concerning the effect of CKD and kidney transplantation over the normal growth rate has increased; now it is known that 40% of children with CKD do not reach the expected height for age. Growth retardation has been associated with the type of nephropathy, metabolic and endocrine disorders that are secondary to kidney disease, immunosuppressive therapy with glucocorticoids, and suboptimal function of renal allograft. Nowadays, we know better the role of the growth hormone/insulin-like growth factor 1 axis in growth retardation we can see it in children with CKD or recipients of renal allograft. Several studies have shown that administration of recombinant growth hormone (rhGH) has a positive effect on the longitudinal growth of children and teenagers who have received a kidney transplant. On the other hand, there have been reported side effects associated with using rhGH; however, these are not statistically significant. In this article, we show a small review about growth in children with CKD and/or recipients of renal allografts the growth pattern of three children who were known by the Transplant Group of National University of Colombia, and the results obtained with the use of rhGH in one of these cases. We want to show the possibility of achieving a secure use of rhGH in children with CKD and its use as a therapeutic option for treating the growth retardation in children with kidney transplantation, and set out the need of typifying the growth pattern of Colombian children with CKD and/or who are recipients of renal allografts through multicenter studies to propose and analyze the inclusion of rhGH in the therapeutic scheme of Colombian children with these two medical conditions. rhGH could be a useful tool for treating children with CKD or kidney transplantation who have not reached the expected longitudinal growth for age. However, it is necessary to know the growth pattern standards for Colombian children with CKD or kidney transplant in Bogotá-Colombia to include the rhGH in clinical protocols for treatment of these patients.

Preventive effects of chronic exogenous growth hormone levels on diet-induced hepatic steatosis in rats.

BackgroundNon-alcoholic fatty liver disease (NAFLD), which is characterized by hepatic steatosis, can be reversed by early treatment. Several case reports have indicated that the administration of recombinant growth hormone (GH) could improve fatty liver in GH-deficient patients. Here, we investigated whether chronic exogenous GH levels could improve hepatic steatosis induced by a high-fat diet in rats, and explored the underlying mechanisms.ResultsHigh-fat diet-fed rats developed abdominal obesity, fatty liver and insulin resistance. Chronic exogenous GH improved fatty liver, by reversing dyslipidaemia, fat accumulation and insulin resistance. Exogenous GH also reduced serum tumour necrosis factor-alpha (TNF-alpha) levels, and ameliorated hepatic lipid peroxidation and oxidative stress. Hepatic fat deposition was also reduced by exogenous GH levels, as was the expression of adipocyte-derived adipokines (adiponectin, leptin and resistin), which might improve lipid metabolism and hepatic steatosis. Exogenous GH seems to improve fatty liver by reducing fat weight, improving insulin sensitivity and correcting oxidative stress, which may be achieved through phosphorylation or dephosphorylation of a group of signal transducers and activators of hepatic signal transduction pathways.ConclusionsChronic exogenous GH has positive effects on fatty liver and may be a potential clinical application in the prevention or reversal of fatty liver. However, chronic secretion of exogenous GH, even at a low level, may increase serum glucose and insulin levels in rats fed a standard diet, and thus increase the risk of insulin resistance.

Evidence for a decrease in cardiovascular risk factors following recombinant growth hormone administration in abstinent anabolic-androgenic steroid users

Objectives To determine whether six days recombinant human growth hormone (rhGH) in an abstinent anabolic-androgenic steroid (AAS) group had any cardiovascular and biochemical effects compared with a control group. Methods Male subjects ( n = 48) were randomly divided, using a single blind procedure into two groups: (1) control group (C) n = 24, mean ± SD, age 32 ± 11 years; height 1.8 ± 0.06 m; (2) rhGH using group (0.058 IU kg −1 day −1) (GH) n = 24, mean ± SD, age 32 ± 9 years; height 1.8 ± 0.07 m. Physiological responses, anthropometry, arterial pulse wave velocity (APWV), blood pressure (BP), heart rate (HR), peak oxygen uptake (VO 2 peak) and biochemical indices were investigated. Results Body mass index, fat-free mass index and VO 2 peak significantly increased while body fat significantly decreased within GH (all P < 0.017). Insulin like growth factor-I significantly increased within GH ( P < 0.017) and compared with C ( P < 0.05). Serum sodium significantly increased ( P < 0.017) and serum homocysteine, high sensitivity C-reactive protein, thyroid stimulating hormone and tetra-iodothyronine (T 4), significantly decreased within GH (all P < 0.017). T 4 significantly decreased compared with C ( P < 0.05). Arterial pulse wave velocity, peak and recovery systolic and diastolic BP, significantly decreased compared with C ( P < 0.05). Resting HR and rate pressure product (RPP) significantly increased compared with C ( P < 0.05). Conclusion The findings of this study suggest that short term use of rhGH may have beneficial effects on endothelial function and specific inflammatory markers of cardiovascular disease in abstinent AAS users, but may have an adverse effect on the cardiovascular system, as evidenced by the increase in resting RPP.

Dangers of growth hormone therapy in critically ill patients.

Prolonged length of stay is the major challenge for modern intensive care because of the associated morbidity and the impact on resource utilization. Severe trauma or infection is associated with a catabolic response, which is characterized by increased protein turnover and negative nitrogen balance. Severe catabolism leads to end-organ dysfunction and muscular weakness, prolonging the need for mechanical ventilation. Catabolism cannot be prevented with standard parenteral or enteral nutritional formulas. In order to prevent the complications of catabolism in intensive care patients, recombinant growth hormone has been applied as an experimental therapy for two decades in patients requiring parenteral nutrition and in patients with respiratory failure. Administration of recombinant growth hormone has resulted in positive nitrogen balance, and studies in mechanically ventilated patients suggest that it may shorten the need for ventilatory support. In contrast to the results of these relatively small studies, a recent multinational randomized controlled trial revealed that the administration of recombinant growth hormone (with doses 10-20 times higher than used for replacement therapy) increases mortality of critically ill patients. The excessive mortality in patients treated with recombinant growth hormone was related to infections and development of multiple organ failure, leading to the conclusion that administration of high doses of recombinant growth hormone cannot be recommended for critically ill patients. This review reinforces that conclusion.

Management of catabolism in metabolically stressed patients: a literature survey about growth hormone application.

In the effort to improve the long-term outcome in critically ill patients, the utilization of anabolic agents, such as human recombinant growth hormone, has been proposed in order to reduce catabolism and improve nutritional status. A recent multicentre study regarding the use of human recombinant growth hormone in intensive care unit patients showed an unexpected increase in the mortality rate in human recombinant growth hormone-treated patients. This finding is in contrast with previous literature data reporting either no differences or an even lower mortality rate with the administration of human recombinant growth hormone. This review evaluates the possible reasons for this dramatic difference in outcomes between the multicentre study and the existing literature. Articles dealing with human recombinant growth hormone administration either in intensive care unit patients (n=26) or in postoperative patients (n=16) have been reviewed. Our analysis suggests that the low caloric intake given to patients enrolled in the multicentre study might have been inadequate to compensate for the hypermetabolism of these patients, and could not support the prolonged and delayed administration of high doses of human recombinant growth hormone. Whether the beneficial metabolic effects of human recombinant growth hormone translate into better clinical outcomes deserves further investigation. In addition, the careful selection of patients to be treated, and close monitoring of both the adequacy of caloric support and modality of human recombinant growth hormone administration would favour the safety of human recombinant growth hormone utilization in critical care settings.

Growth Hormone: A Promising Treatment for the Failing Heart?

Many pathologic processes that accelerate the progression of heart failure, such as cardiac remodeling and impaired contractility, may be modulated by administration of recombinant growth hormone. The agent improves structural and functional aspects of the failing heart both in the short term and after several months of therapy. However, conflicting clinical results cast doubt on whether it has a clear benefit in all of these patients. In addition, growth hormone therapy may be associated with cardiac and noncardiac adverse effects. Many questions must be addressed before its place in heart failure therapy is established. Optimal patient population, dosing regimen, duration of therapy, and effect on patient survival are unknown. Until larger, blinded studies are completed, growth hormone therapy remains an investigational approach to managing refractory heart failure.

The Effects of Bovine Recombinant Growth Hormone Administration on Insulin-like Growth Factor-I and the Haemopoietic System in Thoroughbred Geldings

The Effects of Bovine Recombinant Growth Hormone Administration on Insulin-like Growth Factor-I and the Haemopoietic System in Thoroughbred Geldings

The Effects of Bovine Recombinant Growth Hormone Administration on Insulin-like Growth Factor-I and the Haemopoietic System in Thoroughbred Geldings

SUMMARYThe effect of intramuscularly administered recombinant bovine growth hormone (rbGH) on insulin-like growth factor-I (IGF-I) and white and red blood cell indices was studied in Thoroughbred geldings. An insulin-like growth factor binding protein (IGFBP)-blocked radioimmunoassay was modified and validated for the measurement of IGF-I in equine blood plasma. Baseline values of IGF-I and blood indices were determined over a 48 h period and then a single dose of 5 μg/kg, 10 μg/kg or 50 μg/kg of rbGH was administered. Insulin-like growth factor-I levels increased in a dose-dependent manner, with the highest values between 12 h and 24 h. The highest dose (50 μg/kg) yielded the greatest IGF-I response with a 90.2 ± 10.8% increase at 24 h. White blood cell count increased following the three doses of rbGH with the highest white blood cell count at 12 h after the 50 μg/kg dose. Haemoglobin was significantly increased at 24 h (P< 0.05), when values following doses of 10 μg/kg and 50 μg/kg were significantly greater than after the vehicle or the dose of 5 μg/kg. Red blood cell count was not affected by any of the rbGH doses. These results indicated that rbGH is biologically active in the horse and that rbGH at a dose rate of 10 μg/kg or more could be used therapeutically.

Time course of the response to recombinant growth hormone in acidotic mice.

Long-term administration of recombinant growth hormone (GH) increases growth, despite persistent acidosis, in mice with carbonic anhydrase II deficiency (CAD). In contrast, short-term administration of GH to acidotic rats fails to improve growth. To determine the factors affecting the rate of growth response to GH in acidosis, we analyzed serial measurements obtained during a randomized trial of GH in CAD mice and healthy controls. Administration of GH increased standardized growth progressively up to 5-9 weeks. These data suggest that assessment of the response to GH in rodents requires prolonged administration.

Nutritional aspects in hemodialysis

The results of cross sectional studies throughout the world indicate that maintenance hemodialysis patients are at risk of malnutrition. Longitudinal studies show that malnutrition is associated with a reduced life expectancy mainly because of cardiovascular and infectious complications. Several factors are responsible for malnutrition of hemodialysis patients. Protein-energy intake is often reduced because of inappropriate dietary restrictions, anorexia, and taste alterations, promoting malnutrition in most patients entering dialysis. Intercurrent illnesses and frequent hospitalizations add to meal disturbances. A state of persistent catabolism may result from acidosis, resistance to anabolic factors such as growth hormone, insulin, and insulin-like growth factor-1, as well as a chronic inflammatory state caused by dialysis membrane and fluid bioincompatibility. In addition, losses of nutrients, including glucose, amino acids, proteins, and vitamins, occur during the dialysis treatment. Careful monitoring of dietary intakes is mandatory even in predialysis patients. In hemodialysis patients, the dose of dialysis should be adapted to correct acidosis and to relieve anorexia caused by accumulation of uremic toxins and hyperleptinemia. When malnutrition is established, active therapeutic interventions should take place, including intradialytic parenteral nutrition if oral supplementation has failed to improve nutritional status. Anabolism has been observed during the administration of recombinant growth hormone and insulin-like growth factor-1. Emerging therapeutic strategies against malnutrition may also involve a short period of daily dialysis.

Test method: GH

The abuse of recombinant human growth hormone in sports is considered to be a widespread phenomenon, but until today all our knowledge thereof is based on rumor, anonymous surveys or accidental observations by customs officers. Although growth hormone (GH) is listed as a prohibited class E substance by the International Olympic Committee (IOC) no official test for the detection of GH abuse is implemented. Because of the high degree of identity in the amino acid sequence between recombinant and endogenous GH and because of the pulsatile nature of GH secretion, as well as inter-individual variations, it was believed to be undetectable. Recently, new proposals have been made to overcome this problem. On the basis of differential immunoassays it is possible to determine the relative content of GH isoforms in the circulation and thus to detect the application of recombinant GH. Furthermore, changes in GH dependent parameters after the administration of recombinant GH have been shown to be a possible indicator of GH abuse.

Disturbances of growth hormone-insulin-like growth factor axis and response to growth hormone in acidosis.

Severe chronic metabolic acidosis (CMA) in rats is associated with poor food intake and downregulation of growth hormone (GH), insulin-like growth factors (IGFs), and liver receptors; the administration of recombinant GH (rGH) fails to improve the growth failure. In mice with carbonic anhydrase II deficiency (CAD), a model of moderate CMA with food intake close to normal, we studied serum levels of GH, IGFs, and IGF-binding proteins, and the growth response to rGH. CAD was associated with low serum levels of GH in males. Randomized administration of rGH from approximately 5 to approximately 12 wk to CAD mice improved food efficiency and increased serum IGF-I levels, final length, and weight compared with placebo without affecting blood pH. Although administration of rGH also increased linear growth in healthy animals, the effect was less than that in CAD mice and was only observed when started before 6 wk of life. Thus growth failure in CAD mice is associated with a decrease in GH secretion in males but not in females. Long-term administration of rGH increases linear growth in CAD mice despite persistent CMA.