hGH Concentration Research Articles

Concentration levels of selected hormones in judokas and the extent of their changes during a special performance test at different ambient temperatures

BackgroundThere is little scientific literature available on the diversity of physiological responses of judokas to anaerobic interval exercises in warm environments. Understanding the dynamics of changes in the concentration of selected hormones during a special endurance test at different ambient temperatures may have significant practical value, as it provides an opportunity for optimal programming and monitoring of the training process. So, the main aim of the research was to survey interval anaerobic exercises in different ambient temperatures on Concentration levels of selected hormones in judokas.Methods15 judokas athletes (age: 20.65 ± 2.03 years; body height: 178.00 ± 6.31 cm; body mass: 76.26 ± 12.57 kg; training experience: 12.1 ± 1.57 years) volunteered for the study. The judokas performed five sequences (each lasting 7.20 min) of pulsatile exercises on a cycle ergometer and hand ergometer in a thermoclimatic chamber at temperatures of 21 ± 0.5 °C and 31 ± 0.5 °C. The exercises were different from typical interval exercises, with varying times, upper and lower limb loads, and were followed by a 15-minute break after each sequence. Total duration of the experiment, including the five sequences of pulsating exercise and four 15-minute rest breaks between each exercise sequence, amounted to 96 min and 20 s. The workload was increased by 20 W for the lower limb tests and 12 W for the upper limb tests every 2 min. Biochemical measurements of testosterone (T), cortisol (C), growth hormone (HGH), adrenocorticotropic hormone (ACTH), follicle-stimulating hormone (FSH), adrenaline (E), noradrenaline (NE), and β-endorphin (β-end)were performed using the enzyme-linked immunosorbent assay (ELISA) method on blood samples taken before and after five series of pulsatile exercises, at 1, 24, and 48 h.ResultsPulsatile exercise at ambient temperatures of 21 and 31 °C resulted in a decrease in body weight of the studied athletes (p < 0.05) and significantly reduced body volume and plasma volume after training (p < 0.05). The concentration of HGH, testosterone, cortisol and NE showed a statistically significant difference after the end of the series of pulsating exercises at both temperatures (p < 0.05) and did not significantly affect the concentration of ACTH, FSH and adrenaline concentration.ConclusionsAn increase in the concentration of growth hormone, cortisol and NE was observed after doing the work at both 21 and 31 °C ambient temperature. Physical exertion in both ambient temperatures contributed to a statistically significant decrease in testosterone concentration. Based on the obtained research results, it can be concluded that physical activity in various thermal conditions of the external environment activates the hormonal response to varying degrees, with the direction of changes depending on the external thermal factor.

Neuropsychological effects and cognitive deficits associated with exposure to mercury and arsenic in children and adolescents of the Mojana region, Colombia

The present study aims to explore the neuropsychological performance and exposure to mercury (Hg) and arsenic (As) in Colombian children and adolescents considering biomarkers in hair and blood. The total sample consisted of 70 participants from the Mojana region, Colombia. A neuropsychological evaluation protocol was used, consisting of 11 internationally recognized tests, with evidence of national validation and use for measurement in neurotoxicology contexts. A 57.1% of the sample presented levels above the reference value according to the USEPA, the WHO, and the ATSDR for total mercury in hair (HgH), blood mercury (HgB), and/or blood arsenic (AsB). The mean values reported for HgH were 1.76 ± 3.1 μg/g (95% CI 1.02–2.50) with ranges between 0.23 and 17.20; for HgB: 4.11 ± 5.93 μg/l (CI 2.69–5.52), ranging between 0.25 and 25.80, and for AsB: 1.96 ± 2.73 (CI 1.31–2.61) ranging between 0.50 and 15.50. In the comparison of groups in relation to the level of exposure, a significant difference was found (p < 0.05) for the subtest The Boston Naming Test (BNT). Correlation analyses found, on the one hand, significant negative relationships in tests integrated within the verbal (CVB) and executive function (EF) components as well as evidence of positive relationships in the manifestation of errors in their performance with the levels of Hg and/or As transformed by biomarker (Log10). In the linear regression analyses it was found that for each increase in the concentration of HgH, HgB, and AsB it is estimated that, both for the models with and without data adjustment, there is a loss of scores in integrated measures within the CVB and EF for the transformed levels of HgH, HgB, and AsB; and an increase in the reported errors in their processing within these functions. According to these results, exposure to mercury and/or arsenic is related with performance in verbal neuropsychological skills and executive functioning.

Magnetically actuating implantable pump for the on-demand and needle-free administration of human growth hormone

A bolus of human growth hormone (hGH) is often prescribed for the treatment of growth hormone deficiency, which requires frequent injections in current clinical settings. This painful needle-involved delivery often results in poor patient compliance, leading to low medication adherence and poor clinical outcomes. Therefore, we propose a magnetically actuating implantable pump (MAP) that can infuse an accurate dose of hGH only at the time of non-invasive magnet application from the skin. The MAP herein could reproducibly infuse 20.6 ± 0.9 μg hGH per actuation without any leak at times without actuation. The infused amount increased proportionally with an increase in the number of actuations. When the MAP was implanted and actuated with a magnet in animals with growth hormone deficiency for 21 days, the profiles of plasma hGH concentration and insulin-like growth factor (IGF)-1, as well as changes in body weight, were similar to those observed in animals treated with conventional subcutaneous hGH injections. Therefore, we anticipate that the MAP fabricated in this study can be a non-invasive alternative to administer hGH without repeated and frequent needle injections.

Magneto-Immunoassay for the Detection and Quantification of Human Growth Hormone.

Physiological and endocrine maintenance of a normal human growth hormone (hGH) concentration is crucial for growth, development, and a number of essential biological processes. In this study, we describe the preparation and characterization of magnetic nanoparticles coated with a gold shell (MNPs-Au). The optimal surface concentration of monoclonal anti-hGH antibodies (m-anti-hGH) on magnetic nanoparticles, as well as conditions that decrease non-specific interactions during the magneto-immunoassay, were elaborated. After the selective recognition, separation, and pre-concentration of hGH by MNPs-Au/m-anti-hGH and the hGH interaction with specific polyclonal biotin-labeled antibodies (p-anti-hHG-B) and streptavidin modified horseradish peroxidase (S-HRP), the MNPs-Au/m-anti-hGH/hGH/p-anti-hGH-B/S-HRP immunoconjugate was formed. The concentration of hGH was determined after the addition of 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide substrate solution for HRP; the absorbance at 450 nm was registered after the addition of STOP solution. The developed sandwich-type colorimetric magneto-immunoassay is characterized by a clinically relevant linear range (from 0.1 to 5.0 nmol L−1, R2 0.9831), low limit of detection (0.082 nmol L−1), and negligible non-specific binding of other antibodies or S-HRP. The obtained results demonstrate the applicability of the developed magneto-immunoassay for the concentration and determination of hGH in the serum. Additionally, important technical solutions for the development of the sandwich-type colorimetric magneto-immunoassay are discussed.

In-vitro Release Evaluation of Growth Hormone from an Injectable In-Situ Forming Gel Using PCL-PEG-PCL Thermosensitive Triblock.

An injectable long acting In-Situ Forming Gel (ISFG) of human Growth Hormone (hGH) was prepared by using triblock PCL-PEG-PCL (Mw 1500-1500-1500). Ring-Opening Polymerization (ROP) of triblock using microwave was applied. The BCA protein assay Kit was used to determine the concentration of hGH in the in-vitro release medium. Finally, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) tests and Circular Dichroism (CD) spectrum were done to approve the stability of released hGH. The result of ROP demonstrated that the proportion of PCL to PEG accorded with the initial molar ratio of the monomers. The cross-section of the Surface Electron Microscopy (SEM) indicated the porous framework of the hydrogel could load the drug into its tridimensional matrixes structure. There is the low initial burst release of hGH from the supramolecular hydrogel. The maximum in-vitro release of hGH was 71.2 % ± 1.5 that were due to hGH degrading after this time (21 days). The CD spectrum and SDS-PAGE results confirmed the stability of hGH during invitro release evaluation. The results suggest that the sustained-release formulation using PCL-PEG-PCL can be applied to control the release of hGH.

Evaluation of Sorbitol-Methanol Co-Feeding Strategy on Production of Recombinant Human Growth Hormone in Pichia Pastoris.

Recombinant protein production in Pichia pastoris is based on alcohol oxidase promoters which are regulated by methanol. However, the use of methanol has several disadvantages, which is why current trends in bioprocess development with Pichia pastoris (P. pastoris) are focusing on methanol mixed feeding strategies. This work aimed to develop a new experimental method and compare the effect of various fractions of sorbitol in mixed feeding strategy with stepwise addition of methanol to maximize the productivity of human growth hormone. Accordingly, fed-batch culture performed with a mixed feed of sorbitol/methanol. Sorbitol at concentrations of 30, 40, 50 and 60 gram per liter was added in batch-wise mode to the medium at the beginning of induction phase and the rate of methanol addition was increased stepwise during the first 12 h of production and then kept constant. In order to understand the effects of various co-substrate feeding strategies on P. pastoris and its production of hGH, cell mass, dry cell weight, total protein, hGH expression level and hGH concentration were analyzed and the results compared with the basic feeding protocol using one-way analysis of variance (ANOVA). According to the obtained results, sorbitol at a concentration of 50 g/L could significantly increase the production yield. Under such optimal conditions cell biomass was 108 g/L (DCW), total protein was 0.807 g/L, expression level was 83.1% and rhGH concentration was 0.667 g/L following 30 h induction.

Acute effects of heated resistance exercise in female and male power athletes.

To determine the effects of heated resistance exercise on thermal strain, neuromuscular function and hormonal responses in power athletes. Sixteen (n=8 female; 8 male) highly trained power athletes completed a combined strength and power resistance exercise session in hot (HOT ~30°C) and temperate (CON ~20°C) conditions. Human growth hormone (hGH), cortisol and testosterone concentrations in plasma, peak power (counter-movement jump, CMJ) and peak force (isometric mid-thigh pull) were measured before and after each training session; thermoregulatory responses were monitored during training. Skin temperature, thermal sensation and thermal discomfort were higher in HOT compared with CON. Sweat rate was higher in HOT for males only. Compared with CON, HOT had trivial effects on core temperature and heart rate. During HOT, there was a possible increase in upper-body power (medicine ball throw) in females [3.4% (90% CL -1.5, 8.6)] and males [(3.3% (-0.1, 6.9)], while lower-body power (vertical jump) was enhanced in males only [3.2% (-0.4, 6.9)]. Following HOT, CMJ peak power [4.4% (2.5; 6.3)] and strength [8.2% (3.1, 13.6)] were enhanced in female athletes, compared with CON, while effects in males were unclear. Plasma hGH concentration increased in females [83% (18; 183)] and males [107% (-21; 444)] in HOT compared with CON, whereas differential changes occurred for cortisol and testosterone. Heated resistance exercise enhanced power and increased plasma hGH concentration in female and males power athletes. Further research is required to assess the ergogenic potential of resistance exercise in the heat.

Sulfur-based absolute quantification of proteins using isotope dilution inductively coupled plasma mass spectrometry

An element-based reductive approach provides an effective means of realizing International System of Units (SI) traceability for high-purity biological standards. Here, we develop an absolute protein quantification method using double isotope dilution (ID) inductively coupled plasma mass spectrometry (ICP-MS) combined with microwave-assisted acid digestion for the first time. We validated the method and applied it to certify the candidate protein certified reference material (CRM) of human growth hormone (hGH). The concentration of hGH was determined by analysing the total amount of sulfur in hGH. Next, the size-exclusion chromatography method was used with ICP-MS to characterize and quantify sulfur-containing impurities. By subtracting the contribution of sulfur-containing impurities from the total sulfur content in the hGH CRM, we obtained a SI-traceable certification value. The quantification result obtained with the present method based on sulfur analysis was in excellent agreement with the result determined via a well-established protein quantification method based on amino acid analysis using conventional acid hydrolysis combined with an ID liquid chromatography-tandem mass spectrometry. The element-based protein quantification method developed here can be generally used for SI-traceable absolute quantification of proteins, especially pure-protein standards.

Cloning, expression, and purification of a synthetic human growth hormone in Escherichia coli using response surface methodology.

The aim of this study was to achieve high-level production of the human growth hormone (hGH) in the prokaryotic expression system. In this regard, we performed cloning, expression, and purification of a synthetic hGH gene in BL21 (DE3) strain of E. coli. The hGH production was determined by SDS-PAGE and western blotting techniques, and then the protein concentration was determined by the Bradford assay. To gain insight into the effect of different nutrients on the growth of E. coli and hGH production, in a preliminary assessment nine different types of the basal medium were analyzed. The highest growth of E. coli and hGH production were observed in TB and SOB media. Accordingly, design of experiments was employed for screening the most significant nutrients, and central composite face design was applied for the optimization. The optimum medium consisted of yeast extract (10 g/L), tryptone (10 g/L), and K2HPO4 (2 g/L). The optimum hGH concentration was 391 mg/L, which was 3-fold higher than the hGH concentration in the LB basal medium (119 mg/L). This production rate is the highest hGH concentration reported in the IPTG-inducible expression systems.

Specific and nonspecific interactions in ultraweak protein-protein associations revealed by solvent paramagnetic relaxation enhancements.

Weak and transient protein–protein interactions underlie numerous biological processes. However, the location of the interaction sites of the specific complexes and the effect of transient, nonspecific protein–protein interactions often remain elusive. We have investigated the weak self-association of human growth hormone (hGH, KD = 0.90 ± 0.03 mM) at neutral pH by the paramagnetic relaxation enhancement (PRE) of the amide protons induced by the soluble paramagnetic relaxation agent, gadodiamide (Gd(DTPA-BMA)). Primarily, it was found that the PREs are in agreement with the general Hwang-Freed model for relaxation by translational diffusion (J. Chem. Phys.1975, 63, 4017–4025), only if crowding effects on the diffusion in the protein solution are taken into account. Second, by measuring the PREs of the amide protons at increasing hGH concentrations and a constant concentration of the relaxation agent, it is shown that a distinction can be made between residues that are affected only by transient, nonspecific protein–protein interactions and residues that are involved in specific protein–protein associations. Thus, the PREs of the former residues increase linearly with the hGH concentration in the entire concentration range because of a reduction of the diffusion caused by the transient, nonspecific protein–protein interactions, while the PREs of the latter residues increase only at the lower hGH concentrations but decrease at the higher concentrations because of specific protein–protein associations that impede the access of gadodiamide to the residues of the interaction surface. Finally, it is found that the ultraweak aggregation of hGH involves several interaction sites that are located in patches covering a large part of the protein surface.

Analysis of urinary human growth hormone (hGH) using hydrogel nanoparticles and isoform differential immunoassays after short recombinant hGH treatment: Preliminary results

Successful application clinical-grade human growth hormone (hGH) immunoassays to the discovery of illegal doping cases has been rare. Indeed, the preferred biological matrix in doping control is urine, where the estimated baseline concentration of hGH falls well below the linear range and sensitivity threshold of all commercially available immunoassays, including hGH isoform differential immunoassays which can discriminate pituitary endogenous hGH from recombinant hGH. We employed hydrogel nanoparticles as a pre-processing step that concentrate urinary hGH into the linear range of isoform differential immunoassays.We explored the characteristics of immunoassays in urine spiked with both phGH or rhGH, after pre-treatment with the nanoparticles. Subsequently, pre-treatment was applied to urine obtained from 3 healthy volunteers administered during three days with daily subcutaneous injections of 0.026mg/kg/day rhGH, Genotonorm®.Linearity between both rhGH and phGH concentrations in urine measured by a chemoluminescent assay (Immulite) and in the particle eluate was evident for differential immunoassays (R square higher than 0.999). In case of treated individuals the recombinant/pituitary concentration ratios remained above the established World Anti-Doping Agency (WADA) criterion for hGH misuse up to 24h after the last administration dose, using both assays for volunteer 1 and 2 while in case of volunteer 3 results were inconclusive.The use of nanoparticles appears to open the possibility of assessing rhGH misuse in urine.

Tracking growth hormone abuse in sport: A comparison of distinct isoform-based assays

Detecting recombinant human growth hormone (rhGH) abuse in sport remains one of the major challenges in doping control. We have compared two different approaches to detect the hGH (human growth hormone) abuse. The first measures the concentrations of the 22kDa hGH isoform (rec assay) and pituitary derived isoforms (pit assay) and a ratio rec/pit is obtained. The second measures the concentrations of 22 and 20kDa hGH isoforms and also a ratio 22/20kDa is derived.Using a single set (nine healthy male subjects, 7 days, 0.026mg/kg/day of rhGH, 2 week wash out period) both approaches were compared. To quantify the agreement between the immunoassays, B.A. (Bland–Altman) analysis and P.r. (Pearson correlation) were used. To fully understand the assay readings, all relevant antibodies were characterised by surface plasmon resonance (SPR).In either approach the ratio numerator produces similar results and the denominator determines both signal-amplitude and time-frame of possible application. The rec vs pit approach displays a higher distinctive capacity to detect hGH abuse but the complex binding properties of the capture antibodies make it very difficult to evaluate the precise contributions of the individual hGH variants to the assay result. In the 22 vs 20 approach, the 20kDa hGH concentration measures determine its applicability.Both approaches are based on a different principle, should be preferably applied within 24h after rhGH administration, and are perfectly comparable given the results obtained. The reduced time frame of application indicates that their principle application should be preferably in an out-of-competition setting.

Octreotide Lar Affects the Volume of Pituitary Adenoma in Acromegalic Patients

We studied changes of pituitary adenoma volumes in patients treated with octreotide LAR (SSLAR) over 12 months prior to surgery. 26 patients (22 female and 4 male, mean age 57.5±15.0 years) were treated with SSLAR: 5 patients with microadenoma - during 6 months, and 21 patients with macroadenoma - during 12 months before surgery. Concentration of hGH and IGF-1 was evaluated at 0, 3, 6 and 12 months, while MRI images were taken at 0, 6 and 12 months prior to surgery. The volume of pituitary adenoma, estimated as that of an ellipsoid, was based on MRI image projections. Basic statistics, Shapiro-Wilk and Wilcoxon tests were applied. Median values of hGH and IGF-1 concentrations prior to treatment were 19.0 ng/ml (IQR=40.7) and 766.4 ng/ml (IQR=787.7), respectively. After 6 and 12 months the median value of hGH concentration decreased to 6.3 ng/ml (IQR=9.0) and 3.45 ng/ml (IQR=3.7), respectively, while the median values of IGF-1 became 535.0 ng/ml (IQR=652.8) and 287.0 (IQR=275.7), respectively. All differences were statistically significant (p<0.05) as compared to basal value. The median volumes of adenomas differed significantly and were estimated at: 1.1 cm (3) (IQR=2.5) prior to treatment, 0.5 cm (3) (IQR=1.5) after 6 months, and 0.35 cm (3) (IQR=2.4) after 12 months of SSLAR administration. Treatment of acromegalic patients with somatostatin analogues not only decreases the concentration of hGH and IGF-1, but also appears to decrease the size of the tumour in about 50% of patients treated, leading to general enhancement of the outcome of surgery.

OR12,67 Age-dependent control of Akt phosphorylation in muscles from IGFBP-2 transgenic mice as a potential target for the metabolic effects of IGFBP-2

proliferation assays, the dose-response effect induced by the human growth hormone mutants did not differ from the effects induced by wildtype hGH. Besides, wildtype hGH and the four hGH variants were able to activate STAT5 to the same extent. However, it was observed that production and secretion of the human growth hormone variants were significantly lower than wildtype hGH, as determined by fluorescent sandwich-type immunoassays and confirmed with Western blot analysis. Particularly low secretion was assessed for P89L and V110F, which showed only 15% and 24% of wildtype hGH concentration, respectively. Co-expression of wildtype and mutant human growth hormone furthermore suggested inhibition of wildtype hGH production by mutant hGH. These results indicate that the short stature in patients with one of the four heterozygous missense mutations might be caused by an intracellular event within the somatotropic cells in the anterior pituitary leading to reduced production and/or secretion of the hGH variants as well as of wildtype hGH.

Controlled release of protein drugs from newly developed amphiphilic polymer-based microparticles composed of nanoparticles

A novel formulation of biodegradable microparticles was developed for the sustained release of peptide and protein drugs. The microparticles were formed by the aggregation of protein nanoparticles through water-in-oil (W/O) emulsion-lyophilization and subsequent solid-in-oil-in-water (S/O/W) emulsion–solvent evaporation. Amphiphilic copolymers were used as an emulsifier in the W/O emulsion and matrix of the microparticles. Among the various copolymers investigated, poly(lactide-co-glycolide)-grafted dextran (Dex-g-PLGA) was chosen as the best candidate on the basis of the encapsulation efficiency and in vitro release profile, the near zero-order release without a significant initial burst, of human growth hormone (hGH). The release rate of hGH was controllable by changing the composition of Dex-g-PLGA. The in vivo release studies using normal mice revealed that the plasma concentration of hGH was maintained for 1 week without a significant initial burst. The enhancement of biological activity of hGH by sustained release was confirmed by measuring the IGF-1 concentration and body weight of hypophysectomized mice. These results suggest the high potential of the newly developed microparticles for the sustained release of biopharmaceuticals.

Nanoparticle technology: amplifying the effective sensitivity of biomarker detection to create a urine test for hGH

Several clinical-grade immunoassays exist for the specific measurement of hGH or its isoforms in blood but there is an urgent need to apply these same reliable assays to the measurement of hGH in urine as a preferred 'non-invasive' biofluid. Unfortunately, conventional hGH immunoassays cannot attain the sensitivity required to detect the low concentrations of hGH in urine. The lowest limit of sensitivity for existing hGH immunoassays is >50 pg/mL, while the estimated concentration of urinary hGH is about 1 pg/m-50 times lower than the sensitivity threshold. We have created novel N-isopropylacrylamide (NIPAm)-based hydrogel nanoparticles functionalized with an affinity bait. When introduced into an analyte-containing solution, the nanoparticles can perform, in one step, (1) complete harvesting of all solution phase target analytes, (2) full protection of the captured analyte from degradation and (3) sequestration of the analyte, effectively increasing the analyte concentration up to a hundredfold. N-isopropylacrylamide nanoparticles functionalized with Cibacron Blue F3GA bait have been applied to raise the concentration of urinary hGH into the linear range of clinical grade immunoassays. This technology now provides an opportunity to evaluate the concentration of hGH in urine with high precision and accuracy.

Clonidine (Haemiton) as a reliable stimulus of growth hormone secretion in patients with short stature.

In 14 patients with short stature aged 4-14 years the effect of clonidine has been investigated. A single oral dose of 0.15 mg/m2 surface area is followed by a marked increase of hGH concentration in blood with a peak value between 60 and 90 min after the administration of the drug. The hGH increase caused by clonidine was comparable to the rise during insulin-induced hypoglycemia. In 5 patients both stimuli failed to increase hGH release. The test procedure is simple. It shows only minimum side effects and can therefore be especially recommended in outpatients.

Bioreaction network flux analysis for human protein producing Bacillus subtilis based on genome-scale model

To interpret the effect of human protein gene, e.g., human growth hormone gene hGH, on the intracellular bioreaction network of Bacillus subtilis, the intracellular reaction fluxes were calculated by solving the mass-balance-based genome-scale mathematical model, at pseudo-steady state by using bioreactor data. The bioreaction network consists of 1340 reactions including 990 metabolites. Reaction fluxes in B. subtilis carrying pMK4 ::pre(subC)::hGH (rBsHGH) and B. subtilis carrying merely pMK4 (rBsP) were calculated to identify the existence of pathways and to elucidate the physiological state of the cells. Considering the characteristic cell and recombinant hGH (rhGH) concentration profiles of rBsHGH, the bioprocess was divided into five periods. The reaction network of rBsHGH started operation with 328 reactions (Period I: 0< t<4 h) and lowered to 313 in Period II (4< t<12 h), increased to 314 in Period III (12< t<16 h) by starting rhGH synthesis, and decreased to 296 reactions in Period IV (16< t<24 h) where cell and rhGH formation proceeded; further, decreased to 135 reactions in Period V (24< t<32 h) where cell formation ends and rhGH synthesis continues. However, the reaction network of rBsP started operation with 322 reactions, and lowered to 318 in Period II, and decreased to 43 reactions in Period III where cell formation ends. The flux of the reaction R1340 toward rhGH was the highest (2.0×10 −4 mmol g −1DW h −1) in Period V. The foreign protein gene stimulated and prolonged the cell synthesis (R1339) till the end of Period IV. To increase rhGH production further an exponential glucose feeding strategy between Periods III–V to maintain the specific growth rate μ within 0.02–0.08 h −1, is proposed.

Concentration and preservation of very low abundance biomarkers in urine, such as human growth hormone (hGH), by Cibacron Blue F3G-A loaded hydrogel particles

Urine is a potential source of diagnostic biomarkers for detection of diseases, and is a very attractive means of non-invasive biospecimen collection. Nonetheless, proteomic measurement in urine is very challenging because diagnostic biomarkers exist in very low concentration (usually below the sensitivity of common immunoassays) and may be subject to rapid degradation. Hydrogel nanoparticles functionalized with Cibacron Blue F3G-A (CB) have been applied to address these challenges for urine biomarker measurement. We chose one of the most difficult low abundance, but medically relevant, hormones in the urine: human growth hormone (hGH). The normal range of hGH in serum is 1 to 10 ng/mL but the urine concentration is suspected to be a thousand times less, well below the detection limit (50 pg/mL) of sensitive clinical hGH immunoassays. We demonstrate that CB particles can capture, preserve and concentrate hGH in urine at physiological salt and urea concentrations, so that hGH can be measured in the linear range of a clinical immunometric assay. Recombinant and cadaveric hGH were captured from synthetic and human urine, concentrated and measured with an Immulite chemiluminescent immunoassay. Values of hGH less than 0.05 ng/mL (the Immulite detection limit) were concentrated to 2 ng/mL, with a urine volume of 1 mL. Dose response studies using 10 mL of urine demonstrated that the concentration of hGH in the particle eluate was linearly dependent on the concentration of hGH in the starting solution, and that all hGH was removed from solution. Thus if the starting urine volume is 100 mL, the detection limit will be 0.1 pg/mL. Urine from a healthy donor whose serum hGH concentration was 1.34 ng/mL was studied in order detect endogenous hGH. Starting from a volume of 33 mL, the particle eluate had an hGH concentration of 58 pg/mL, giving an estimated initial concentration of hGH in urine of 0.175 pg/mL. The nanotechnology described here appears to have the desired precision, accuracy and sensitivity to support large scale clinical studies of urine hGH levels.

Human growth hormone measurement by means of a sensitive ELISA of whole blood spots on filter paper

Background Measurements of human growth hormone (hGH) are a prerequisite for identifying a deficiency or excess. Our study is the first to investigate the reliability of a very sensitive assay for the quantification of GH in dried blood spots on filter paper. Objective Validation of a commercially-available enzyme-linked immunoassay (ELISA) for measuring hGH from filter paper samples of dried blood. Methods We used an assay system (ELISA, E022, Mediagnost) based on polyclonal rabbit antibodies. Its suitability is ascribable to its very high sensitivity (1.6 ng/L) and virtual absence of interfering factors, excepting for a cross-reactivity with high pegvisomant concentrations. Results hGH was found to be stable in dried blood spots on filter paper (No. 903, Whatman) over eight days at 37 °C. Extraction of hGH from filter paper, in comparison to EDTA plasma, was 107% (SD 8.1%; n = 6) over a range from 2.4 to 34.5 μg/L. Linear regression analysis ( n = 119) showed a correlation of R 2 = 0.97 for the hGH concentration in serum and on filter paper samples. Conclusion Our findings demonstrate the reliability of measurements of hGH in dried blood spots on filter paper. The advantages of this method are the low sample volume and the easy transport, storage, and handling of samples. This method contributes to the standardisation of diagnostics pertaining to abnormal hGH secretion as it facilitates the comparison of decisive measurements.