Short stature explained by dimerization of human growth hormone induced by a p.C53S point mutation.

A homozygous mutation in growth hormone 1 (GH1) was recently identified in an individual with growth failure. This mutation, c.705G>C, causes replacement of cysteine at position 53 of the 191-amino-acid sequence of 22 kDa human GH (hGH) with serine (p.C53S). This hGH molecule (hereafter referred to as GH-C53S) lacks the disulfide bond between p.Cys-53 and p.Cys-165, which is highly conserved among species. It has been reported previously that monomeric GH-C53S has reduced bioactivity compared with WT GH (GH-WT) because of its decreased ability to bind and activate the GH receptor in vitro In this study, we discovered that substitution of p.Cys-53 in hGH significantly increased formation of hGH dimers in pituitary cells. We expressed His-tagged hGH variants in the cytoplasm of genetically modified Rosetta-gami B DE3 Escherichia coli cells, facilitating high-yield production. We observed that the bioactivity of monomeric GH-C53S is 25.2% of that of GH-WT and that dimeric GH-C53S-His has no significant bioactivity in cell proliferation assays. We also found that the expression of GH-C53S in pituitary cells deviates from that of GH-WT. GH-C53S was exclusively stained in the Golgi apparatus, and no secretory granules formed for this variant, impairing its stimulated release. In summary, the unpaired Cys-165 in GH-C53S forms a disulfide bond linking two hGH molecules in pituitary cells. We conclude that the GH-C53S dimer is inactive and responsible for the growth failure in the affected individual.

Circulating aryl hydrocarbon receptor-interacting protein (AIP) is independent of GH secretion.

BACKGROUND: Aryl hydrocarbon receptor-interacting protein (AIP) is evolutionarily conserved and expressed widely throughout the organism. Loss-of-function AIP mutations predispose to young-onset pituitary adenomas. AIP co-localizes with growth hormone in normal and tumorous somatotroph secretory vesicles. AIP protein is detectable in circulation. We aimed to investigate possible AIP and GH co-secretion, by studying serum AIP and GH levels at baseline and after GH stimulation or suppression, in GH deficiency (GHD) and in acromegaly patients. SUBJECTS AND METHODS: Insulin tolerance test (ITT) was performed in GHD patients (n = 13) and age-BMI-matched normal GH axis control patients (n = 31). Oral glucose tolerance test (OGTT) was performed in active acromegaly patients (n = 26) and age-BMI-matched normal GH axis control patients (n = 18). In-house immunometric assay was developed for measuring circulating AIP. RESULTS: Serum AIP levels were in the 0.1 ng/mL range independently of gender, age or BMI. Baseline AIP did not differ between GHD and non-GHD or between acromegaly and patients with no acromegaly. There was no change in peak, trough or area under the curve during OGTT or ITT. Serum AIP did not correlate with GH during ITT or OGTT. CONCLUSIONS: Human circulating serum AIP in vivo was assessed by a novel immunometric assay. AIP levels were independent of age, sex or BMI and unaffected by hypoglycaemia or hyperglycaemia. Despite co-localization in secretory vesicles, AIP and GH did not correlate at baseline or during GH stimulation or suppression tests. A platform of reliable serum AIP measurement is established for further research of its circulatory source, role and impact.

Human placental growth hormone in ectopic pregnancy: Detection in maternal blood, immunohistochemistry and potential clinical implication.

OBJECTIVE: To investigate human placental growth hormone (hGH-V) in ectopic pregnancy (EP): detection in maternal blood, correlation with immunohistochemistry and possible role as a marker for the course of EP. DESIGN: Women presenting in the outpatient or emergency department of a tertiary care university hospital with a positive pregnancy test and strong suspicion of EP by ultrasound and/or symptoms were eligible for the study (n=70). Tissue specimens from the surgically treated patients (n=50) were examined by histopathology as well as by a hGH-V specific immohistochemistry set-up. A highly sensitive hGH-V specific immunoassay was used to analyse serum samples collected before treatment, day 1 post surgery samples and serial samples for medical treatment. RESULT(S): In EP patients' sera hGH-V was shown to be measurable for the first time (n=18). HGH-V however could not be detected in all patients' sera. HCG levels were significantly higher in the hGH-V serum positive group (p 0.001). HGH-V was localized to the syncytiotrophoblast in all specimens of EP examined by immunohistochemistry (n=10) regardless of the detection in the patient's blood. CONCLUSION(S): Placental growth hormone (hGH-V) was shown to be present both in ectopic pregnancy patients' sera and tissue. It may serve as a biomarker for monitoring the course and treatment of EP.

Sex-specific and inter-individual differences in biomarkers of selenium status identified by a calibrated ELISA for selenoprotein P.

Selenoprotein P (SELENOP) is a liver-derived transporter of selenium (Se) in blood, and a meaningful biomarker of Se status. Se is an essential trace element for the biosynthesis of enzymatically-active selenoproteins, protecting the organism from oxidative damage. The usage of uncalibrated assays hinders the comparability of SELENOP concentrations and their pathophysiological interpretation across different clinical studies. On this account, we established a new sandwich SELENOP-ELISA and calibrated against a standard reference material (SRM1950). The ELISA displays a wide working range (11.6-538.4µg/L), high accuracy (2.9%) and good precision (9.3%). To verify whether SELENOP correlates to total Se and to SELENOP-bound Se, serum samples from healthy subjects and age-selected participants from the Berlin Aging Study II were analyzed by SELENOP-ELISA and Se quantification. SELENOP was affinity-purified and its Se content was determined from a subset of samples. There was a high correlation of total Se and SELENOP concentrations in young and elderly men, and in elderly women, but not in young women, indicating a specific sexual dimorphism in these biomarkers of Se status in young subjects. The Se content of isolated SELENOP was independent of sex and age (mean±SD: 5.4±0.5). By using this calibrated SELENOP-ELISA, prior reports on pathological SELENOP concentrations in diabetes and obesity are challenged as the reported values are outside reasonable limits. Biomarkers of Se status in clinical research need to be measured by validated assays in order to avoid erroneous data and incorrect interpretations, especially when analyzing young women. The Se content of circulating SELENOP differs between individuals and may provide some important diagnostic information on Se metabolism and status.

Human placental growth hormone: a potential new biomarker in gestational trophoblastic disease.

OBJECTIVES: Gestational trophoblastic disease (GTD) involves a spectrum of abnormal proliferations arising from the placental villous trophoblast. Although the incidence is low, a biomarker with short serum half-life would be a major clinical advance to monitor surgical and medical treatment reducing the socioeconomic burden of multiple control visits as well as patient's anxiety. Placental growth hormone (hGH-V) plays an important role in the regulation of normal placental growth and has shown angiogenic effects. We aimed to determine by immunohistochemistry (IHC) whether hGH-V is expressed in GTD and whether it can be detected in the patient's blood for potential monitoring of surgical or medical treatment procedures. METHODS: Tissue and sera were collected from women undergoing treatment for GTD in a tertiary care university hospital. We evaluated partial and complete hydatidiform moles, invasive moles and choriocarcinoma, n=16. Trophoblast specimens were examined by a newly developed IHC set-up for hGH-V in addition to gross morphologic and histopathological examination. Serum samples were analyzed by a highly sensitive hGH-V specific immunoassay. RESULTS: hGH-V was localized in all entities of GTD to the syncytiotrophoblast by immunohistochemistry. Serum hGH-V was detected for the first time in GTD and was present in a high percentage of all analyzed entities. CONCLUSIONS: hGH-V can be detected in all entities of GTD by IHC as well as by serum analysis and may therefore serve as a novel biomarker for the disease. Its clinical utility in diagnosis of GTD and monitoring surgical or medical treatment needs to be determined in further studies.

Detection of 3,5-diiodothyronine in sera of patients with altered thyroid status using a new monoclonal antibody-based chemiluminescence immunoassay.

BACKGROUND: 3,5-Diiodo-L-thyronine (3,5-T2), a potential metabolite of 3,3',5-triiodothyronine (T3), exerts marked metabolic actions without the undesirable cardiac and central side effects of T3. So far the lack of reliable quantification methods for endogenous 3,5-T2 in human serum has limited further insight into its physiological and pathophysiological roles in endocrine homeostasis and disease status. METHODS: Monoclonal anti-3,5-T2 antibodies (3,5-T2 mAbs) were produced in mice. We developed a competitive chemiluminescence immunoassay (CLIA) with one selected mAb and optimized it for high sensitivity, linearity, recovery, and low cross-reactivity to structurally related thyroid hormones (THs) and thyronamines. The CLIA was then used to investigate the origin and action of 3,5-T2 in humans under physiological and pathophysiological conditions in comparison with THs. Patient analysis included individuals with confirmed hypo- or hyperthyroidism and a separate population of thyroidectomized patients on L-thyroxine (T4) replacement therapy. RESULTS: 3,5-T2 is stable in human serum after storage at 4°C or room temperature as well as several freeze-thaw cycles. The immunoassay did not show any significant cross-reactivity with naturally occurring TH metabolites in physiological and pathophysiological concentrations. The assay shows a lower detection limit of 0.2 nM 3,5-T2 and an upper detection limit of 10.0 nM. The newly established CLIA generates reliable results after spiking exogenous 3,5-T2 or by linear dilution of sera. Intra-assay variation is between 4.1% and 9.0%. Overall mean of variation between different assays is 5.6%-12.9%. 3,5-T2 serum concentrations do not differ in hyperthyroid (0.31 ± 0.02 nM, n=24) compared to hypothyroid (0.43 ± 0.04 nM, n=31) individuals. 3,5-T2 was detectable and elevated in serum from thyroidectomized and T4-substituted patients (0.48 ± 0.03 nM, n=100) in comparison to a sex- and age-matched control group (0.29 ± 0.01 nM, n=99). CONCLUSION: The established CLIA is highly specific, sensitive, precise and accurate for 3,5-T2 detection in human serum. Because 3,5-T2 is not regulated in conditions of an altered thyroid state, it is most likely that serum 3,5-T2 concentrations are not directly dependent on feedback regulation via the hypothalamic-pituitary axis. In addition 3,5-T2 is present in thyroidectomized individuals on T4 substitution, and it is elevated after T4 substitution compared with healthy controls. We conclude that these data support extrathyroidal production of 3,5-T2 from T4.

Automated 22-kD growth hormone-specific assay without interference from Pegvisomant.

BACKGROUND: Large variability exists among different growth hormone (GH) assays owing to differences in calibration, antibody specificity, isoform recognition, and interference from GH binding protein (GHBP). The GH receptor antagonist Pegvisomant presents a new challenge because Pegvisomant interferes with many GH assays. A recent consensus conference established criteria for standardization and evaluation of GH assays. Following consensus recommendations, we developed a new GH assay on an automated analyzer (IDS-iSYS, Immunodiagnostic Systems). METHODS: A monoclonal antibody not cross-reacting with Pegvisomant was combined with a monoclonal antibody specific for 22-kD GH. Isoform specificity and interference from GHBP was tested and compared to that seen in 2 existing automated GH assays (Siemens Immulite, Diasorin Liaison). We also compared GH concentrations measured by the 3 assays for healthy volunteers and patients with acromegaly receiving different treatments. Using the iSYS assay, we also established nadir GH values during oral glucose load and analyzed changes in endogenous GH during Pegvisomant treatment. RESULTS: Analytical and functional sensitivities were 0.01 µg/L and 0.04 µg/L, with a dynamic range from 0.04 to 100 µg/L. Intraassay CVs were 2%-4%, whereas interassay CVs were 5%-7% at GH concentrations between 1.7 and 27.5 µg/L. The assay was specific for 22-kD GH and not affected by GHBP. The presence of Pegvisomant, which leads to a negative bias on the Immulite and dramatic overestimation of GH on the Liaison, had no impact on the iSYS GH assay. CONCLUSIONS: The new assay fulfils recent consensus recommendations and presents a useful new tool for reliable measurement of GH.

Structure of the human obesity receptor leptin-binding domain reveals the mechanism of leptin antagonism by a monoclonal antibody.

Leptin regulates energy homeostasis, fertility, and the immune system, making it an important drug target. However, due to a complete lack of structural data for the obesity receptor (ObR), leptin's mechanism of receptor activation remains poorly understood. We have crystallized the Fab fragment of a leptin-blocking monoclonal antibody (9F8), both in its uncomplexed state and bound to the leptin-binding domain (LBD) of human ObR. We describe the structure of the LBD-9F8 Fab complex and the conformational changes in 9F8 associated with LBD binding. A molecular model of the putative leptin-LBD complex reveals that 9F8 Fab blocks leptin binding through only a small (10%) overlap in their binding sites, and that leptin binding is likely to involve an induced fit mechanism. This crystal structure of the leptin-binding domain of the obesity receptor will facilitate the design of therapeutics to modulate leptin signaling.

Effects of GH in women with abdominal adiposity: a 6-month randomized, double-blind, placebo-controlled trial.

OBJECTIVE: Abdominal adiposity is associated with increased cardiovascular risk and decreased GH secretion. The objective of our study was to determine the effects of GH on body composition and cardiovascular risk markers in abdominally obese women. MATERIALS AND METHODS: In this randomized, double-blind, placebo-controlled study, 79 obese premenopausal women received GH vs placebo for 6 months. Primary endpoints were i) total abdominal (total abdominal adipose tissue, TAT) fat by computed tomography (CT) (body composition) and ii) high-sensitivity C-reactive protein (hsCRP) (cardiovascular risk marker). Body composition was assessed by CT, dual-energy X-ray absorptiometry, and proton MR spectroscopy. Serum cardiovascular risk markers, carotid intima-media thickness, and endothelial function were measured. RESULTS: Mean 6-month GH dose was 1.7±0.1 mg/day, resulting in a mean IGF1 SDS increase from -1.7±0.08 to -0.1±0.3 in the GH group. GH administration decreased TAT and hsCRP compared with placebo. In addition, it increased thigh muscle mass and lean body mass and decreased subcutaneous abdominal and trunk fat, tissue plasminogen activator, apoB, and apoB/low-density lipoprotein compared with placebo. Visceral adipose tissue (VAT) decreased and intramyocellular lipid increased within the GH group. Six-month change in IGF1 levels was negatively associated with 6-month decrease in TAT and VAT. One subject had a 2 h glucose >200 mg/ml at 3 months; four subjects, three of whom were randomized to GH, had 2 h glucose levels >200 mg/ml at the end of the study. CONCLUSION: GH administration in abdominally obese premenopausal women exerts beneficial effects on body composition and cardiovascular risk markers but is associated with a decrease in glucose tolerance in a minority of women.

Evidence for extrathyroidal formation of 3-iodothyronamine in humans as provided by a novel monoclonal antibody-based chemiluminescent serum immunoassay.

CONTEXT: Thyronamines are thyronergic metabolites of thyroid hormones. Lack of reliable and sensitive detection methods for endogenous 3-iodothyronamine (3-T(1)AM) has so far hampered progress in understanding their physiological action and role in endocrine homeostasis or pathophysiology of diseases. OBJECTIVE: We characterized newly generated mouse monoclonal 3-T(1)AM antibodies and established a monoclonal antibody-based chemiluminescence immunoassay as a powerful tool for monitoring 3-T(1)AM levels in investigations addressing altered serum profiles and potential sites of origin and action of 3-T(1)AM in humans. DESIGN AND SETTING: Our exploratory study on 3-T(1)AM serum levels in humans measured 3-T(1)AM concentrations in comparison with thyroid hormones. PATIENTS OR OTHER PARTICIPANTS: Thirteen adult healthy subjects, 10 patients with pituitary insufficiency, and 105 thyroid cancer patients participated. INTERVENTIONS: INTERVENTIONS included l-T(4) withdrawal in patients with pituitary insufficiency as well as TSH-suppressive T(4) substitution in thyroid cancer patients. RESULTS: 3-T(1)AM was reliably quantified in human serum and stable after storage at room temperature and 4 C overnight as well as after four freeze-thaw cycles. The median serum concentration in healthy subjects was 66 ± 26 nm. 3-T(1)AM was also detected in T(4)-substituted thyroid cancer patients. Although free T(4) and T(3) significantly decreased during T(4) withdrawal, 3-T(1)AM levels remained constant for 6 d. CONCLUSION: Because higher 3-T(1)AM levels are detectable in T(4)-substituted thyroid cancer patients after thyroidectomy/radioiodine treatment compared with healthy controls, we concluded that 3-T(1)AM is mainly produced by extrathyroidal tissues. The serum profile during T(4) withdrawal suggests either a long half-life or persisting 3-T(1)AM release into serum from intracellular thyroid hormone precursors or stores.

Lack of dietary carbohydrates induces hepatic growth hormone (GH) resistance in rats.

GH is a well established regulator of growth, lipid, and glucose metabolism and therefore important for fuel utilization. However, little is known about the effects of macronutrients on the GH/IGF system. We used low-carbohydrate/high-fat diets (LC-HFD) as a model to study the impact of fat, protein, and carbohydrates on the GH/IGF-axis; 12-wk-old Wistar rats were fed either regular chow, a moderate, protein-matched LC-HFD, or a ketogenic LC-HFD (percentage of fat/protein/carbohydrates: chow, 16.7/19/64.3; LC-HF-1, 78.7/19.1/2.2; LC-HF-2, 92.8/5.5/1.7). After 4 wk, body and tibia length, lean body mass, and fat pad weights were measured. Furthermore, we investigated the effects of LC-HFD on 1) secretion of GH and GH-dependent factors, 2) expression and signaling of components of the GH/IGF system in liver and muscle, and 3) hypothalamic and pituitary regulation of GH release. Serum concentrations of IGF-I, IGF binding protein-1, and IGF binding protein-3 were lower with LC-HF-1 and LC-HF-2 (P < 0.01). Both LC-HFD-reduced hepatic GH receptor mRNA and protein expression, decreased basal levels of total and phosphorylated Janus kinase/signal transducers and activators of transcription signaling proteins and reduced hepatic IGF-I gene expression. Hypothalamic somatostatin expression was reduced only with LC-HF-1, leading to increased pituitary GH secretion, higher IGF-I gene expression, and activation of IGF-dependent signaling pathways in skeletal muscle. In contrast, despite severely reduced IGF-I concentrations, GH secretion did not increase with LC-HF-2 diet. In conclusion, lack of carbohydrates in LC-HFD induces hepatic GH resistance. Furthermore, central feedback mechanisms of the GH/IGF system are impaired with extreme, ketogenic LC-HFD.

A microsphere-based duplex competitive immunoassay for the simultaneous measurements of aldosterone and testosterone in small sample volumes: validation in human and mouse plasma.

BACKGROUND: The small blood volumes available in rodent studies often limit adequate quantification of all hormones of interest. We report here the development of two new assays combining an extraction step with multiplex immunoassay (MIA) technology for the simultaneous determination of aldosterone and testosterone in 50 µl sample volume. METHODS: Following solvent extraction, aldosterone and testosterone competitive immunoassays are performed incorporating biotinylated tracers and antibody-coated beads each having a unique fluorescence. Quantification is via addition of streptavidin-R-phycoerythrin (SA-PE). The assays were validated and compared to established methods. Baseline hormone levels in mice from four different strains, and changes after ACTH and HCG stimulation in CD-1 mice are shown. RESULTS: The assays are sensitive (aldosterone 15 pg/ml, testosterone 12 pg/ml), reproducible (intra-/inter-assay imprecision aldosterone 5.1-15.6%/9.9-15.8% and testosterone 9.7-10.9%/7.7-11.4%) and correlate significantly to established assays (r=0.94-0.95). Baseline aldosterone levels varied between strains, but not between the genders. Testosterone was significantly higher in male of all strains except in C57BL/6 × NMRI mice. After ACTH injection, aldosterone (median, interquartile range) rose from 354 (261-396) pg/ml to 2008 (875-2467) in male and from 260 (210-576) to 1120 (734-1528) in female CD-1 mice. HCG injection in the same strain increased testosterone in male mice only (3.5 (0.4-8.3) ng/ml to 31.8 (30.4-33.9) ng/ml, P<0.01). CONCLUSIONS: We describe a MIA for the simultaneous measurement of aldosterone and testosterone in small volumes after extraction. In addition to presenting a new tool for steroid research in rodent models, our data show strain-dependent differences in steroid hormone metabolism in rodents.

Serum levels of 20K-hGH and 22K-hGH isoforms in acromegalic patients.

The diagnosis and treatment decisions in acromegaly depend on the measurement of growth hormone (GH) and insulin-like growth factor I (IGF-I) levels. The occurrence of different GH isoforms in the serum, mainly 22K-hGH and 20K-hGH, is a source of heterogeneity of GH results measured by different immunoassays. Since it has been previously reported that the proportion of 20K-hGH is increased in patients with active acromegaly, it might be useful to know the GH isoforms' pattern not to underdiagnose or undertreat acromegalic patients.

Immunogenicity, toxicology, pharmacokinetics and pharmacodynamics of growth hormone ligand-receptor fusions.

A fundamental concern for all new biological therapeutics is the possibility of inducing an immune response. We have recently demonstrated that an LR-fusion (ligand-receptor fusion) of growth hormone generates a potent long-acting agonist; however, the immunogenicity and toxicity of these molecules have not been tested. To address these issues, we have designed molecules with low potential as immunogens and undertaken immunogenicity and toxicology studies in Macaca fascicularis and pharmacokinetic and pharmacodynamic studies in rats. Two variants of the LR-fusion, one with a flexible linker (GH-LRv2) and the other without (GH-LRv3), were tested. Comparison was made with native human GH (growth hormone). GH-LRv2 and GH-LRv3 demonstrated similar pharmacokinetics in rats, showing reduced clearance compared with native GH and potent agonist activity with respect to body weight gain in a hypophysectomized rat model. In M. fascicularis, a low level of antibodies to GH-LRv2 was found in one sample, but there was no other evidence of any immunogenic response to the other fusion protein. There were no toxic effects and specifically no changes in histology at injection sites after two repeated administrations. The pharmacokinetic profiles in monkeys confirmed long half-lives for both GH-LRv2 and GH-LRv3 representing exceptionally delayed clearance over rhGH (recombinant human GH). The results suggest that repeated administration of a GH LR-fusion is safe, non-toxic, and the pharmacokinetic profile suggests that two to three weekly administrations is a potential therapeutic regimen for humans.

Specific monoclonal antibodies and ultrasensitive immunoassays for 20K and 22K human growth hormone.

OBJECTIVE: Generation of specific monoclonal antibodies (mAbs) against 20K and 22K human growth hormone (hGH) and development of ultrasensitive immunoassays to quantify 20K and 22K hGH. DESIGN: Mice were immunized with recombinant 20K or 22K hGH. Hybridoma cells were screened with biotinylated 20K and 22K hGH simultaneously. The specific mAbs were further characterized and used for construction of isoform specific assays. The ultrasensitive chemiluminescent assays were developed with AMDEX streptavidin-HRP and a sensitive substrate. RESULTS: The 20K hGH specific mAb 1G12 and the 22K hGH specific mAb 5E1 showed less than 0.1% cross-reactivity to 22K or 20K hGH by competitive binding assay, respectively. Western blot analysis also confirmed the specificity of mAb 1G12 and mAb 5E1. Using mAb 1G12 and mAb 5E1, 20K and 22K specific assays with working range of 2-2000 pg/mL were constructed. The 22K hGH concentrations in 103 serum samples from different healthy subjects in the basal GH state were 343.7+/-421.5 pg/mL (18.6-1820 pg/mL). The 20K hGH concentrations were 30.7+/-37.5 pg/mL (2.4-205pg/mL). The ratios of 20K to 20K plus 22K hGH were 9.8+/-4.4% (3.3-28.3%). Both 22K hGH and 20K hGH concentrations in women (465.9+/-476.3 pg/mL and 43.7+/-46.1 pg/mL, n=47) were significantly higher than those (241.1+/-337.0 pg/mL and 20K hGH 19.8+/-23.0 pg/mL, n=56, P<0.01) in men. However, there was no difference in the proportion of 20K to 20K plus 22K between men and women (P>0.05). The strong correlation between 20K and 22K hGH (R=0.914, P<0.01) indicated the constant proportion between 20K and 22K hGH in the basal GH state of healthy subjects. CONCLUSIONS: Specific monoclonal antibodies and ultrasensitive chemiluminescent immunoassays for 20K and 22K hGH were generated. The ultrasensitive immunoassays are essential for the determination of 20K and 22K hGH in the basal GH state. This universal ultrasensitive immunoassay form can be adapted to other immunoassays for broad application.

IGF-1, IGFBP-3 and ALS in adult patients with chronic kidney disease.

BACKGROUND: Insulin-like growth factor I (IGF-1) is for the most part bound in a ternary complex with IGF-binding protein-3 (IGFBP-3) and acid-labile subunit (ALS). This ternary complex is a storage form of IGF-1 in blood and passes not through the renal glomerulus. Little information is available in regard to the components of the ternary complex in adult renal disease. OBJECTIVE: To investigate levels of serum IGF-1, IGFBP-3 and ALS in relation to renal function and extent of proteinuria. DESIGN AND PATIENTS: We measured IGF-1, IGFBP-3 and ALS concentrations in 137 patients who were investigated due to proteinuria and/or haematuria and/or renal impairment. The patients received renal biopsies and the histological diagnosis was documented. Urinary albumin excretion and relevant clinical parameter were evaluated. RESULTS: IGF-1 showed a highly positive correlation to IGFBP-3 and ALS, and the latter to IGFBP-3. IGF-1, IGFBP-3 and ALS decreased with increasing age. IGF-1 and IGFBP-3 showed no significant change depending on the creatinine clearance. However, ALS decreased with decreasing renal function. In patients with heavy proteinuria ALS levels, but not IGF-1 and IGFBP-3 levels, decreased significantly. Patients with chronic ischaemic renal damage and diabetic glomerulopathy showed higher IGF-1 and IGFBP-3 levels compared to patients with thin glomerular basement membrane disease despite their older age. CONCLUSIONS: IGF-1 and IGFBP-3 levels seem to be independent of renal function and severity of proteinuria. However, ALS levels are altered in renal failure and nephrotic syndrome, which may be due to increased renal loss or diminished hepatic production or both.

Growth hormone isoforms in acromegalic patients before and after treatment with octreotide LAR.

BACKGROUND: Human growth hormone (hGH) circulates as a mixture of different isoforms. It has been previously reported that the ratio of 20kDa to 20kDa plus 22kDa (%20kDa-hGH) is increased in patients with active acromegaly. OBJECTIVES: To evaluate the GH isoforms (20kDa- and 22kDa-hGH) in acromegalic patients before and after six months of treatment with octreotide LAR, and to compare the results with those in healthy controls. In addition, the relationships between the %20kDa-hGH, tumor size and biochemical measurements were also investigated. DESIGN: Random serum samples from 23 acromegalic patients evaluated before and after six months of treatment with octreotide LAR and from 23 matched healthy controls were studied. Growth hormone, IGF-I and prolactin (PRL) were measured by chemiluminescence immunometric assay and the 20kDa- and 22kDa-hGH isoforms were measured by specific time-resolved fluorescence immunoassays. RESULTS: In acromegalic patients before treatment, there was a significantly higher median %20Da-hGH in comparison to healthy controls (14.31% vs. 9.59%, p<0.001). After six months of treatment, the median %20kDa-hGH was similar to the baseline values. Patients with GH<2.5ng/mL after six months of treatment had already lower GH and %20kDa-hGH at baseline (p<0.01). The IGF-I (SD-scores) was positively correlated to total GH levels in acromegalic patients after treatment. There was no correlation between the %20kDa-hGH and PRL levels or tumor size. CONCLUSIONS: Our study confirmed that acromegalic patients have an increased proportion of circulating 20kDa-hGH isoform. Consequently, the use of a 22kDa-hGH specific assay may underestimate the tumor production of total GH. Although octreotide LAR promoted a significant decrease in the GH and IGF-I levels, it did not normalize the GH isoforms composition and suggests that the secretion of GH isoforms is equally inhibited by somatostatin analogues and that it is the disease control that normalizes the GH isoforms composition in acromegaly.

High-sensitivity chemiluminescence immunoassays for detection of growth hormone doping in sports.

BACKGROUND: Recombinant human growth hormone (rhGH) is abused in sports, but adequate routine doping tests are lacking. Analysis of serum hGH isoform composition has been shown to be effective in detecting rhGH doping. We developed and validated selective immunoassays for isoform analysis with potential utility for screening and confirmation in doping tests. METHODS: Monoclonal antibodies with preference for pituitary hGH (phGH) or rhGH were used to establish 2 pairs of sandwich-type chemiluminescence assays with differential recognition of rhGH (recA and recB) and phGH (pitA and pitB). We analyzed specimens from volunteers before and after administration of rhGH and calculated ratios between the respective rec- and pit-assay results. RESULTS: Functional sensitivities were <0.05 microg/L, with intra- and interassay imprecision < or =8.4% and < or =13.7%, respectively. In 2 independent cohorts of healthy subjects, rec/pit ratios (median range) were 0.84 (0.09-1.32)/0.81 (0.27-1.21) (recA/pitA) and 0.68 (0.08-1.20)/0.80 (0.25-1.36) (recB/pitB), with no sex difference. In 20 recreational athletes, ratios (median SD) increased after a single injection of rhGH, reaching 350% (73%) (recA/pitA) and 400% (93%) (recB/pitB) of baseline ratios. At a moderate dose (0.033 mg/kg), mean recA/pitA and recB/pitB ratios remained significantly increased for 18 h (men) and 26 h (women). After high-dose rhGH (0.083 mg/kg), mean rec/pit ratios remained increased for 32 h (recA/pitA) and 34 h (recB/pitB) in men and were still increased after 36 h in women. CONCLUSIONS: Using sensitive chemiluminescence assays with preferential recognition of phGH or rhGH, detection of a single injection of rhGH was possible for up to 36 h.

Pegvisomant-primed GH stimulation test.

OBJECTIVE: Provocative stimulation tests for GH assessment have poor reproducibility and can often elicit false positive results in normal children. The aim of our study was to evaluate the capability of pegvisomant, as an enhancer of GH secretion, in unmasking false-positive results in short children undergoing GH testing. DESIGN: A prospective study was conducted between March 2005 and April 2006. PATIENTS: Twenty-one short children (8 males and 13 females), aged 1.0-14.5 years, with a height of < 2 SD scores below the mean were included in the study. METHODS: All subjects underwent an L-DOPA stimulation test with evaluation of GH. At the end of the test, 1 mg/kg of pegvisomant was given subcutaneously and 3 days later an L-DOPA stimulation test was repeated. RESULTS: There was a significant decrease of IGF-I SDS following pegvisomant (-1.75 +/- 0.24 vs.-2.65 +/- 0.23; P < 0.0001) and a significant increase of the GH-peak (6.2 +/- 0.91 vs. 15.3 +/- 2.30 microg/l; P < 0.0001). Among the 21 patients examined, 18 (85.7%) had an insufficient response (< 10 microg/l) at the first stimulation. Ten of them (55.5%) showed normal secretion after priming with pegvisomant, while insufficient secretory reserve was confirmed in the remaining eight. CONCLUSIONS: Pegvisomant priming before GH stimulation tests can be used to improve the reliability of the diagnostic work up in GH deficiency. Further studies are required, however, to clarify whether this procedure should be recommended in the routine evaluation of GH status.

A ligand-receptor fusion of growth hormone forms a dimer and is a potent long-acting agonist.

Cytokine hormones have a short plasma half-life and require frequent administration. For example, growth hormone replacement involves daily injections. In common with other cytokines, the extracellular domain of the growth hormone receptor circulates as a binding protein, which naturally prolongs the biological half-life of growth hormone. Here we have studied the biological actions of a ligand-receptor fusion of growth hormone and the extracellular domain of its receptor. The genetically engineered ligand-receptor fusion protein was purified from mammalian cell culture. In rats, the ligand-receptor fusion had a 300-times reduced clearance as compared to native growth hormone, and a single injection promoted growth for 10 d, far exceeding the growth seen after administration of native growth hormone. The ligand-receptor fusion forms a reciprocal, head-to-tail dimer that provides a reservoir of inactive hormone similar to the natural reservoir of growth hormone and its binding protein. In conclusion, a ligand-receptor fusion of cytokine to its extracellular receptor generates a potent, long-acting agonist with exceptionally slow absorption and elimination. This approach could be easily applied to other cytokines.