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Dilution linearity and correlation analysis with LC-MS/MS measurements. (A) The dilution linearity of the assay with three different native rabbit serum samples in relation to recombinant rabbit IGF-I (r-rbIGF-I) and recombinant human (r-hIGF-I) standard curves. (B) The correlation of rabbit IGF-I measurements obtained using either the immunoassay or LC-MS/MS by using Passing–Bablock regression. Rabbit IGF-I concentrations were on average approximately twofold higher when measured by using LC-MS/MS (Spearman's ρ, 0.813; P<0.0001; Passing–Bablock: slope 0.684 and intercept −78.3). The solid line shows the regression fit; the dotted lines show the 95% confidence limits of the regression fit.
Source publication
The development of new growth hormone (GH) agonists and growth hormone antagonists (GHAs) requires animal models for pre-clinical testing. Ideally, the effects of treatment are monitored using the same pharmacodynamic marker that is later used in clinical practice. However, intact rodents are of limited value for this purpose because serum IGF-I, t...
Contexts in source publication
Context 1
... for measurement of IGF-I using this immunoassay is published elsewhere ( Bidlingmaier et al., 2014). With respect to dilution linearity in native rabbit samples, serial dilutions of the three tested native rabbit samples paralleled the dilution of the recombinant standard material used (i.e. recombinant human IGF-I and recombinant rabbit IGF-I; Fig. ...
Context 2
... that had been made by using immunoassay and liquid chromatography tandem mass spectrometry (LC-MS/MS) revealed a good overall correlation of measurement results, but the rabbit IGF-I concentrations were on average 1.9-fold higher when measured by using LC-MS/MS (Spearman's ρ, 0.813; P<0.0001; Passing-Bablock: slope 0.684 and intercept −78.3; Fig. ...
Context 3
... Serum concentrations of Pegvisomant increased until the fourth injection to reach a maximum circulating concentration of 17990±2107 ng/ml. The GHA concentrations remained at this high level until the last injection was given and then declined again (Fig. 4B). As expected, the GHA was not detectable in the control group. Supplementary material Fig. 1 shows that after a single subcutaneous injection of the GHA, the serum GHA levels steadily increased to reach the highest GHA concentrations after 8 h. In comparison to recombinant human GH, the GHA was cleared much more slowly from the circulation (supplementary material Fig. ...
Context 4
... GHA was not detectable in the control group. Supplementary material Fig. 1 shows that after a single subcutaneous injection of the GHA, the serum GHA levels steadily increased to reach the highest GHA concentrations after 8 h. In comparison to recombinant human GH, the GHA was cleared much more slowly from the circulation (supplementary material Fig. ...
Context 5
... of controls (PBS 305.7±9.4 ng/ml versus recombinant human GH 436.7±15.1ng/ml; P<0.001; Fig. 4C). In contrast to GHA, recombinant human GH displayed a different pharmacodynamic behavior in the circulation. Peak concentrations were detected 8 h after an injection, with rapidly decreasing blood concentrations of recombinant human GH thereafter ( Fig. 4D; supplementary material Fig. S1). IGFBP-2 concentrations in rabbits increased approximately threefold 24 h after being injected with recombinant human GH (P<0.001; Fig. 4E). IGFBP-3 and IGFBP-4 levels were not significantly affected by recombinant human GH (data not shown). With respect to changes in body weight, the recombinant-human-GH group showed the highest ...
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Citations
... In humans, IGF-1 levels are associated with body mass index and fat deposits in adult women [38]. In addition, the IGF-1 gene is widely used in agriculture as a potential biomarker [39,40]. ...
Aquaculture has tremendous economic significance in distinguishing males and females in the juvenile Silurus asotus (Linnaeus, 1758) to obtain a female population with tremendous growth potential. To investigate the potential biological markers between young males and females S. asotus, we analyzed the characteristics of sexual dimorphism by measuring the 14 length traits and 9 weight indicators in an artificial insemination population at 3, 5, and 7 months. In addition, quantitative real-time PCR (qRT-PCR) was performed to determine the sexually dimorphic expression of the growth hormone-1 gene (GH-1), growth hormone receptor gene (GHR), and insulin-like growth factor gene (IGF-1) in the hypothalamus, pituitary, gonad, and liver, at 3, 5, and 7 months. The results showed that in morphology, except for eye diameter and the distance between the pelvic and anal fins in 3-month fish, all other morphological indicators were significantly ( P < 0.05 ) or very significantly ( P < 0.01 ) different between juvenile males and females. The visceral weight, eviscerated weight, and intestine weight in females were significantly ( P < 0.05 ) or very significantly ( P < 0.01 ) higher than in males at 5 and 7 months. Joint static allometric analyses on 14 length indicators relative to weight showed different sex growth patterns in 3-month, 5-month, and 7-month fish. In gene expression patterns, GH-1, IGF-1, and GHR were highly expressed in the pituitary, with higher levels in females ( P < 0.05 or P < 0.01 ). In contrast, the three genes were all more highly expressed in the testis than in the ovary ( P < 0.01 ), indicating their essential roles in testis development. Our results demonstrate that S. asotus has female-biased sexual dimorphism. The length traits related to head shapes could be the potential phenotype marker to distinguish females and males in 7-month juveniles.
... Patients who suffer from GH deficiency, Prader-Willi, and Turner syndrome, receive a daily injection of this hormone (2). So, there is a strong need for new dosage forms that can facilitate hGH delivery, reduce the number of injections, and increase treatment efficacy and patients' compliance (2)(3)(4). However, hGH instability in new drug delivery systems (NDDSs), aqueous solutions remain a major hurdle. ...
Objective:
It is so difficult to formulate human growth hormone (hGH) in a solution with high stability and new drug delivery system (NDDs) due to physiochemical instability. The purpose of this study was to investigate the possibility of using Tris as a hGH stabilizer.
Materials and methods:
In this experimental study, the role of tris(hydroxymethyl)aminomethane (Tris) was evaluated as a hGH stabilizing agent in phosphate buffer, as a practical aqueous solution and a media to release NDDs. Highperformance liquid chromatography (HPLC) and enzyme-linked immune sorbent assay (ELISA) were applied to investigate the stability of hGH in solutions and dynamic light scattering (DLS) was used to measure the effect of Tris on the hydrodynamic size of hGH in aqueous solutions. Ultra violet (UV) spectrophotometry was used to check the hGH spectrum. In computational study, formation of ligand-protein complex of the Tris-hGH, and the intermolecular interactions between Tris and hGH were studied by molecular docking modeling.
Results:
The results demonstrated that Tris at the optimum concentration, increases hGH stability in aqueous solutions. Also, molecular docking modeling confirmed that amino acid residues such as tyrosine (Tyr), proline (Pro), glutamic acid (Glu), aspartic acid (Asp), leucine (Leu), and phenylalanine (Phe) in hGH structure, were linked with Tris as a ligand.
Conclusion:
It seems that interactions between hGH and Tris are the most important reason for increment of the physicochemical stability of hGH in aqueous solutions containing Tris.
... The serum concentration profiles demonstrate a bimodal phenomenon in both B2 and C groups. It is noteworthy that Bielohuby et al. [27] (by IV administration to rabbits) and Kramer et al. [28] observed a redistribution and a small increase but a repetitive pattern (perhaps from muscles). ...
A patient-friendly delivery system to release human growth hormone (hGH) is very desirable. In situ forming implant systems (ISIs) can provide a long acting and effective protein delivery. In these systems, solvents and additives play major roles in drug release. In this study, four groups of PLGA-based ISIs containing hGH were prepared in N-methyl-2-pyrrolidone (NMP) and poly(ethylene glycol) dimethyl ether (PEG-DME) as solvents with and without tris(hydroxymethyl) aminomethane (Tris) as stabilizer. Several analyses were used to investigate the implants, which include release profile, viscosity, contact angle, gel permeation chromatography (GPC), scanning electron microscopy (SEM), hGH and IGF-1 serum measurements and histopathology. In in vitro release experiments, the hGH cumulative release from PEG-DME system was twice that from NMP system during 14 days, and hGH release was tripled in the presence of Tris. With the addition of Tris to the ISIs containing PEG-DME, the water penetration, interconnectivity of pores and inner channels, surface pores and hydrophilicity were increased. Moreover, the effect of Tris on the hGH stabilization synergized its positive effects and increased the hGH final cumulative release. Results of the ISIs containing PEG-DME and Tris injection in rabbits demonstrated a reduced tissue inflammation. Moreover, the 14-days serum levels of the hGH and IGF-1 of this system in recipient rabbits were comparable to those of the commercial daily injection samples.
... As such, no studies have since been published on the 20K GH-V. However, Bielohuby and colleagues report that normal (non-GH deficient) wildtype laboratory rodents have a limited IGF-1 response to exogenous GH treatment despite dramatic changes to glucose homeostasis, body composition, and organ mass (14,15). Bielohuby speculates that IGF-1 release may be near maximum in these animals, making further increases by exogenous GH treatments difficult to achieve (14). ...
... Furthermore, while body length was not significantly increased in their study, Vickers et al did report a trend toward an increased nose-to-tail length (P = 0.06) with GHv treatment, suggesting that an increase in animal numbers may have yielded a significant result (13). Regarding the animal model used, wild-type laboratory rodents have been described by Bielohuby et al to have a limited IGF-1 response to exogenous GH treatment despite changes to glucose homeostasis, body composition, and organ mass (14,15,17). The authors propose that IGF-1 release may be near maximal in wild-type rodents, making further increases by exogenous GH treatments difficult to achieve (14). ...
A rare 20K isoform of GH-V (here abbreviated GHv) was discovered in 1998. To date, only one research article has characterized this isoform in vivo observing that GHv treatment in male high-fat fed rats had several GH-like activities but unlike GH lacked diabetogenic and lactogenic activities and failed to increase IGF-1 or body length. Therefore, the current study was conducted to further characterize the in vivo activities of GHv in a separate species and in a GH deficient model (GH-/- mice) and with both sexes represented. GHv treated GH-/- mice had significant increases to serum IGF-1, femur length, body length, body weight and lean body mass and reduced body fat mass similar to mice receiving GH treatment. GH treatment increased circulating insulin levels and impaired insulin sensitivity, in contrast, both measures were unchanged in GHv treated mice. Since GHv lacks prolactin receptor (PRLR) binding activity, we tested the ability of GH and GHv to stimulate proliferation of human cancer cell lines and found that GHv has a decreased proliferative response in cancers with high PRLR. Our findings demonstrate that GHv can stimulate IGF-1 and subsequent longitudinal body growth in GH deficient mice similar to GH, but unlike GH, GHv promoted growth without inhibiting insulin action and without promoting growth of PRLR-positive cancers in vitro. Thus, GHv may represent improvements to current GH therapies especially for individuals at risk for metabolic syndrome or PRLR-positive cancers.
... During the second half of pregnancy, IGF-I concentration varied between 114.5 to 136.8 ng/ml. According to Bielohuby et al. (2014) who stated that, few studies determined concentration of IGF-I in rabbits and it is need further evaluation to describe the changes in IGF-I level in rabbits. Therefore, circulating IGF-I in rabbits varied widely among studies. ...
... Our current level is comparable to that (149 ng/ml) reported by Costa et al. (1998). In contrast, the present level is much lower than that 371 ng/ml (Thakur et al., 2000) and that 285.8 ng/ml (Bielohuby et al., 2014). Also, our finding completely disagree with that of Alfonso (2016) who stated that, IGF-I level in pregnant rabbits increased gradually with pregnancy advancement recording its lower level (200 ng/ml) at day 14 and the highest levels were at days 21 and 28 (290 and 300 ng/ml, respectively). ...
Pregnancy is a critical period for animals where it undergoes many physiological changes including hormones, which are not received a great attention in rabbit. Therefore, a total number of 25 New Zealand White pregnant rabbit does were used, to assess the changes in concentration of relevant hormones in relation to rabbit productivity. Blood samples were collected on 14, 21 and 28 days of pregnancy and on kindling day to quantify six maternal hormones. Litter size and weight, in addition to average of weekly and total milk yield were determined. Concentrations of the six hormones during the second half of pregnancy ranged between 3.2 to 4.0 ng/ml for progesterone (P4), 47.3 to 89.0 pg/ml for estrogen (E2), 2.0 to 3.7 ng/ml for prolactin (PRL), 114.5 to 136.8 ng/ml for insulin like growth factor-I (IGF-I), 36.0 to 39.2 ng/ml for thyroxine (T4) and 1.9 to 2.2 ng/ml for triiodothyronine (T3). The corresponding values on kindling day were 1.6 ng/ml, 26.8 pg/ml, 4.6 ng/ml, 131.6 ng/ml, 37.4 ng/ml and 0.9 ng/ml, respectively. At day 14, maternal P4, E2, PRL, T4 and T3 were the lowest, whereas IGF-I was the highest compared to the other two days of pregnancy. At day 28, levels of E2, PRL, T4 and T3 were the highest in comparison with days 14 and 21 of pregnancy. On kindling day, P4 and T3 showed the minimal levels, whilst PRL exhibited the maximal level compared to the levels at all the gestational days. The relationship among the different hormones had various trends. The average of total milk yield (129.9 g/d) was negatively correlated with both P4 and E2, and positively associated with the PRL, IGF-I, T4 and T3 hormones. Furthermore, litter size was positively related with P4, E2 and PRL, and negatively with T4, T3 and IGF-I. Whereas, litter weight was negatively correlated with P4, E2 and T4. We recommend giving more attention to the rabbit doe reproduction, particularly close to parturition to achieve good economical return. However, further studies are urgently needed in this area.
... The mean serum IGF-1 concentration has been reported in red deer with the average being 63.6 ng/mL (57.4-79.9 ng/mL; Ditchkoff et al., 2001), and in rabbits as 200 ng/mL (Bielohuby et al., 2014).There are no reports of serum IGF-1 concentration in bovine bulls of breeding age, but in young bulls the IGF-1 concentration ranged from 300 to 400 ng/mL with a gradual increase with age and body weight (Lee et al., 2005;Brito, 2015;Bourgon et al., 2017). In the present study, the IGF-1 concentration of seminal plasma of buffalo bulls (50.34 ± 6.12 ng/mL) was less than that of cattle (116-144 ng/mL; Henricks et al., 1998), but greater than that of stallions (10.2 to 10.4 ng/mL; Macpherson et al., 2002) and boars Fig. 4. Sperm plasma membrane integrity in normal bulls; Values with different letters differ (P < 0.05). ...
The objective of the study was to establish correlation of seminal and serum IGF-1 with seminal attributes, estimate antioxidant potential of IGF-1 by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays and to study the effect of IGF-1 supplementation on semen cryopreservation. For this study, buffalo bulls were divided into sub-fertile (n = 2) and normal (n = 5) on the basis of sperm mass motility and individual motility. The serum IGF-1 concentration of normal bulls was greater than in sub-fertile bulls, but there was no difference in the seminal IGF-1 concentration among the groups. The values from correlation analyses indicated that serum IGF-1 concentration is positively correlated with semen mass motility and sperm concentration. In the second experiment, IGF-1 did not have antioxidant activities when assessed with DPPH and FRAP assays. In the third experiment, the ejaculates of normal and sub-fertile bulls were cryopreserved using semen extender in which there was IGF-1 supplementation at 0 (control), 50, 100, 150, 200, 250, 350 and 450 ng/mL of extender. Supplementation of IGF-1 at 250 ng/ml resulted in improved sperm motility, longevity and membrane intactness as compared to control after cryopreservation of semen from normal buffalo bulls, but not sub-fertile bulls. In summary, serum IGF-1 concentration was correlated with sperm mass motility and concentration in buffalo bulls and supplementation of IGF-1 protected sperm during the cryopreservation process but effects were not due to direct antioxidant activity.
... The mean serum IGF-1 concentration has been reported in red deer with the average being 63.6 ng/mL (57.4-79.9 ng/mL; Ditchkoff et al., 2001), and in rabbits as 200 ng/mL A C C E P T E D M A N U S C R I P T (Bielohuby et al., 2014).There are no reports of serum IGF-1 concentration in bovine bulls of breeding age, but in young bulls the IGF-1 concentration ranged from 300 to 400 ng/mL with a gradual increase with age and body weight (Lee et al., 2005;Brito, 2015;Bourgon et al., 2017). ...
... Important steps of this basic validation include the analysis of precision and linearity; the latter can for example be done with serial dilutions of the calibrators or positive controls which should always be delivered with an immunoassay kit. Other recent examples of hormone-specific and extended, state-of-the-art assay validation efforts for novel immunoassays measuring hormones in samples from laboratory animal species can be found in the cited references (70)(71)(72)(73)(74). In addition, literature research or personal exchange during scientific meetings can be helpful to elucidate if other research groups have successfully used the (potentially novel) immunoassay to measure the analyte. ...
The measurement of circulating hormones by immunoassay remains a cornerstone in preclinical endocrine research. For scientists conducting and interpreting immunoassay measurements of rodent samples, the paramount aim usually is to obtain reliable and meaningful measurement data in order to draw conclusions on biological processes. However, the biological variability between samples is not the only variable affecting the readout of an immunoassay measurement and a considerable amount of unwanted or unintended variability can be quickly introduced during the pre-analytical and analytical phase. This review aims to increase the awareness for the factors "pre-analytical" and "analytical" variability particularly in the context of immunoassay measurement of circulating metabolic hormones in rodent samples. In addition, guidance is provided how to gain control over these variables and how to avoid common pitfalls associated with sample collection, processing, storage and measurement. Furthermore, recommendations are given how to perform a basic validation of novel single- and multiplex immunoassays for the measurement of metabolic hormones in rodents. Finally, practical examples from immunoassay measurements of plasma insulin in mice address the factors "sampling site and inhalation anesthesia" as frequent sources of introducing an unwanted variability during the pre-analytical phases. The knowledge about the influence of both types of variability on the immunoassay measurement of circulating hormones as well as strategies to control these variables are crucial for planning and realization of metabolic rodent studies and for the generation and interpretation of meaningful immunoassay data from rodent samples.
... In order to overcome disadvantages associated with LBAs, MS-based assays have evolved in the recent years as a complementary analytical technology for PK, PD, and IG assessments of mAbrelated therapeutic proteins in complex matrices. [207][208][209][210][211][212][213][214][215][216][217] In contrast to LBAs, MS-based assays offer an increased specificity and robustness, a wider linear dynamic range, shorter method development time, ability to multiplex, and the possibility to implement an ISTD to minimize matrix effects, which facilitates method transfer between biological matrices. [200][201][202][218][219][220][221][222] The majority of MS-based assays utilizes proteolytic peptides as surrogates for an indirect quantification of the parent mAb-related therapeutic protein (bottom-up approach) due to the following reasons: ...
This PhD thesis focused on the development of generic mass spectrometry (MS)-based workflows for monoclonal antibody (mAb)-related therapeutic protein quantification in pre-clinical species. First, the development of bottom-up sample preparation protocols either based on direct serum digestion or immuno-capture allowed mAb-related therapeutic protein quantification over five orders of magnitude whereas the employment of peptides from the constant region of the mAb demonstrated the versatility of such generic liquid chromatography tandem MS (LC-MS/MS)-based approaches. Second, high-resolution MS (HRMS) instruments were evaluated as an alternative to triple quadrupole mass analyzers, traditionally utilized for bottom-up mAb quantification by LC-MS/MS. The major benefit of HRMS incorporation into the workflow was associated with the possibility to quantify simultaneously mAb-related therapeutic proteins directly at an intact level, providing an information level far beyond the one obtained with bottom-up LC-MS/MS methodologies. Hence, the pivotal role of HRMS for the qualitative and quantitative analyses of mAb-related therapeutic proteins was further outlined throughout this doctoral work.
... This method has been shown to be a feasible method to validate potential biomarker candidates especially for complex clinical samples [74][75][76]. An alternative choice is affinity/antibody based methods; Western blotting and enzyme-linked immuno sorbent assay (ELISA) could be used [77][78][79]. ...
Burn injury is a prevalent and traumatic event for pediatric patients. At present, the diagnosis of burn injury severity is subjective and lacks a clinically relevant quantitative measure. This is due in part to a lack of knowledge surrounding the biochemistry of burn injuries and that of blister fluid. A more complete understanding of the blister fluid biochemistry may open new avenues for diagnostic and prognostic development. Burn insult induces a highly complex network of signaling processes and numerous changes within various biochemical systems, which can ultimately be examined using proteome and metabolome measurements. This review reports on the current understanding of burn wound biochemistry and outlines a technical approach for 'omics' profiling of blister fluid from burn wounds of differing severity.
