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Problems with GH assays and strategies toward standardization

Martin Bidlingmaier

Disorders affecting GH secretion – either GH deficiency or GH excess (acromegaly) – are biochemically defined through peak or nadir concentrations of human GH in response to dynamic tests. Immunoassays employing polyclonal or monoclonal antibodies are routinely used for the analysis of GH concentrations, and many different assays are available on the market today. Unfortunately, the actual value reported for the GH concentration in a specific patient's sample to a large extent depends on the assay method used by the respective laboratory. Variability between assay results exceeds 200%, limiting the applicability of consensus guidelines in clinical practice. Reasons for the heterogeneity in GH assay results include the heterogeneity of the analyte itself, the availability of different preparations for calibration, and the interference from matrix components such as GH-binding protein. Furthermore, the reporting of results in mass units or international units together with the application of variable conversion factors led to confusion.

International collaborations proposed measures to improve the comparability of assay results, recommending the use of a single, recombinant calibrator for all assays and reporting only in mass units as first steps. However, because of the differences in epitope specificity of antibodies used in different assays, method-specific cut-off levels for dynamic tests might remain necessary to correctly interpret and compare results from different laboratories.

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Adrenal vein sampling using rapid cortisol assays in primary aldosteronism is useful in centers with low success rates

Matthias J Betz, Christoph Degenhart, Evelyn Fischer, Anna Pallauf, Volker Brand, Ulrich Linsenmaier, Felix Beuschlein, Martin Bidlingmaier, and Martin Reincke

Objective

Adrenal vein sampling (AVS) is considered the gold standard in the differential diagnosis of primary aldosteronism (PA), but success rates vary between centers. We hypothesized that rapid (intraprocedure) cortisol measurement can improve performance in a center with initially low AVS success rate.

Design

We analyzed 46 patients with confirmed PA studied between 2008 and 2010. Forty-seven PA patients studied between 2004 and 2008 identified by retrospective chart review served as controls. All patients were treated at a single tertiary care university hospital.

Methods

Starting in 2008, rapid cortisol assays (RCA) were performed in all patients during the AVS procedure. A cortisol gradient of ≥2.0 between adrenal vein and a femoral vein sample was used as success criterion. Up to two repeat samples were drawn if adrenal vein cortisol was below this threshold.

Results

During the control period 26 of 47 AVS were successful (55%). After introduction of RCA, 39 out of 46 AVS (85%) were successful (P=0.003). In 21 of the 46 cases (46%) a resampling was necessary. The increase in overall success was due to an increase in successful right AVS (85 vs 62% before introduction of RCA; P=0.02) and a training effect (P=0.024 for trend).

Conclusion

RCA during AVS are useful in centers with an initially low AVS success rate.

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Pharmacokinetic and pharmacodynamic profile of a new sustained-release GH formulation, LB03002, in children with GH deficiency

Ferenc Peter, Conrad Savoy, Hyi-Jeong Ji, Mihaly Juhasz, Martin Bidlingmaier, and Paul Saenger

Objective

LB03002 is a novel, sustained-release recombinant human GH, developed for once-a-week s.c. injection. To evaluate the suitability for long-term GH replacement therapy in children with GH deficiency (GHD), the present study assessed the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of LB03002 at three doses.

Study design and patients

The randomised, comparator-controlled, assessor-blinded, phase II study assessed 37 (24 boys, 13 girls) pre-pubertal, GH-naïve children with GHD, in 11 European centres, for PK and PD analyses. GH, IGF1 and IGFBP3 concentrations were measured following the last daily GH dose and the first and 13th once-a-week administration of LB03002 at doses of 0.2, 0.5 or 0.7 mg/kg.

Results

GH C max values after the three doses of LB03002 were increased up to fourfold, with a clear dose proportionality. For each LB03002 dose, GH area under the concentration versus time curve did not increase from the first to 13th (month 3) administration, indicating no accumulation of circulating GH. IGF1 C max showed a progressive increase during LB03002 administration. Conversely, IGFBP3 showed a rapid increase in C max. IGF1 SDS were fully normalised after 3 months of treatment, whereas IGFBP3 SDS were already in the normal range for all the three LB03002 dosages after 1 week.

Conclusions

At the doses used, LB03002 has a suitable profile for long-term treatment to promote growth in children with GHD. The quantitative changes in IGF1 and IGFBP3 indicate adequate stimulation of the IGF system by LB03002 and the pattern of increase is comparable with that seen in GHD children in a standard IGF1 generation test using daily GH.

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Sample pre-treatment determines the clinical usefulness of acid-labile subunit immunoassays in the diagnosis of growth hormone deficiency and acromegaly

Katherine M Morrison, Martin Bidlingmaier, Simone Stadler, Zida Wu, Lars Skriver, and Christian J Strasburger

Objective: The usefulness of measuring the GH-dependent acid-labile subunit (ALS) in the management of GH deficiency (GHD) and acromegaly remains in question and is investigated in this study, comparing several different immunoassays for ALS.

Method: We compared the diagnostic accuracy of a commercially available polyclonal Ab-based ELISA with SDS pre-treatment (SDS-ELISA) with a monoclonal Ab-based immunofluorometric assay, using two unfolding methods (urea (UREA) and Glycine-HCl (Gly)). The corresponding molecular weight (MW) of ALS and IGFBP-3 immunoreactivity was determined. The clinical usefulness of each assay was examined in adult GH disorders.

Results: ALS was lower in GHD and higher in acromegaly using all assays. In GHD, UREA had higher sensitivity and specificity than SDS-ELISA (59 and 69% versus 41 and 51% respectively). In acromegaly, sensitivity and specificity was 94 and 87% for UREA, 81 and 36% for Gly, and 44 and 44% for SDS-ELISA. After UREA, immunoreactivity for ALS and IGFBP-3 eluted at their predicted free MW using size-exclusion chromatography, whereas ALS immunoreactivity in SDS (300–600 kDa) and Gly (250–500 kDa) was at a high apparent MW consistent with aggregation.

Conclusion: The diagnostic accuracy of ALS varies with assay choice and pre-treatment modality. UREA, which results in migration of ALS at the expected MW on a sizing column, has the highest specificity and sensitivity. Thus, if measured in an assay in which ALS is unfolded without aggregation, ALS is a clinically highly useful parameter for the assessment of GH.

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Anthropometric factors have significant influence on the outcome of the GHRH–arginine test: establishment of normative data for an automated immunoassay specifically measuring 22 kDa human growth hormone

Timo Deutschbein, Martin Bidlingmaier, Jochen Schopohl, Christian J Strasburger, and Stephan Petersenn

Context

Adult growth hormone (GH) deficiency (GHD) is diagnosed by provocative testing of GH secretion.

Objective

To improve the diagnostic accuracy of GH-releasing hormone (GHRH) plus arginine (GARG) testing, we evaluated the influence of age, BMI and sex and established normative data for an automatic immunoassay specifically measuring 22 kDa human GH.

Design/setting

Prospective multicenter study.

Participants

Eighty-seven patients with hypothalamic–pituitary disease and 200 healthy controls. Patients were classified according to the number of pituitary hormone deficiencies (PHD). GHD was assumed when ≥2 PHD (in addition to GH) were present (n = 51); 36 patients with <2 PHD were considered GH sufficient (GHS). ROC analysis identified cutoffs with ≥95% specificity for GHD. Controls were prospectively stratified for sex, age and BMI.

Interventions

All participants received GHRH and l-arginine.

Main outcome measures

GH was measured by immunoassay (iSYS, IDS).

Results

In controls, multiple stepwise regression analysis showed that BMI (21%, P < 0.0001), sex (20%, P < 0.0001) and age (5%, P < 0.001), accounted for 46% of GH peak level variability during GARG. Comparison of peak GH during GARG (GHD vs GHS + controls) revealed an overall cutoff of 3.9 ng/mL (sensitivity 86%, specificity 95%). After adjustment for BMI and sex, optimal cutoffs (male vs female) were 6.5 vs 9.7 ng/mL in lean, 3.5 vs 8.5 ng/mL in overweight and 2.2 vs 4.4 ng/mL in obese subjects respectively.

Conclusion

BMI and sex account for most of the variability of peak GH levels during GARG. Consequently, diagnostic accuracy of the GARG test is significantly improved by use of adjusted cutoffs.

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Acute administration of acyl, but not desacyl ghrelin, decreases blood pressure in healthy humans

Cecilia J Zhang, Martin Bidlingmaier, Mekibib Altaye, Laura C Page, David D’Alessio, Matthias H Tschöp, and Jenny Tong

Objective

To compare the effects of acyl ghrelin (AG) and desacyl ghrelin (DAG) on blood pressure (BP), heart rate (HR) and other autonomic parameters in healthy humans and to elucidate the hormonal mechanisms through which AG could exert its cardiovascular effects.

Design

Seventeen healthy participants underwent frequent monitoring of systolic (sBP) and diastolic blood pressure (dBP), HR, respiratory rate (RR) and body surface temperature (Temp) during continuous infusion of AG, DAG, combined AG + DAG or saline control before and during an IV glucose tolerance test on 4 separate days. Plasma catecholamines, renin and aldosterone levels were also measured. Differences in outcome measures between treatment groups were assessed using mixed-model analysis.

Results

Compared to the saline control, AG and combined AG + DAG infusions decreased sBP, dBP, mean arterial blood pressure (MAP), HR and Temp. In contrast, DAG infusion did not alter BP, RR or Temp, but did decrease HR. The AG and AG + DAG infusions also raised plasma aldosterone levels compared to saline (P < 0.001) without affecting renin or catecholamine levels.

Conclusions

The decrease in BP, HR, RR and Temp with AG infusion suggests mediation through the autonomic nervous system. The lack of response to DAG suggests that these autonomic effects require activation of the ghrelin receptor.

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Assessment of lung function in a large cohort of patients with acromegaly

Sylvère Störmann, Bodo Gutt, Josefine Roemmler-Zehrer, Martin Bidlingmaier, Rudolf M Huber, Jochen Schopohl, and Matthias W Angstwurm

Objective

Acromegaly is associated with increased mortality due to respiratory disease. To date, lung function in patients with acromegaly has only been assessed in small studies, with contradicting results. We assessed lung function parameters in a large cohort of patients with acromegaly.

Design

Lung function of acromegaly patients was prospectively assessed using spirometry, blood gas analysis and body plethysmography. Biochemical indicators of acromegaly were assessed through measurement of growth hormone and IGF-I levels. This study was performed at the endocrinology outpatient clinic of a tertiary referral center in Germany.

Methods

We prospectively tested lung function of 109 acromegaly patients (53 male, 56 female; aged 24–82 years; 80 with active acromegaly) without severe acute or chronic pulmonary disease. We compared lung volume, air flow, airway resistance and blood gases to normative data.

Results

Acromegaly patients had greater lung volumes (maximal vital capacity, intra-thoracic gas volume and residual volume: P < 0.001, total lung capacity: P = 0.006) and showed signs of small airway obstruction (reduced maximum expiratory flow when 75% of the forced vital capacity (FVC) has been exhaled: P < 0.001, lesser peak expiratory flow: P = 0.01). There was no significant difference between active and inactive acromegaly. Female patients had significantly altered lung function in terms of subclinical airway obstruction.

Conclusions

In our cross-sectional analysis of lung function in 109 patients with acromegaly, lung volumes were increased compared to healthy controls. Additionally, female patients showed signs of subclinical airway obstruction. There was no difference between patients with active acromegaly compared with patients biochemically in remission.

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Lack of influence of somatic mutations on steroid gradients during adrenal vein sampling in aldosterone-producing adenoma patients

Andrea Oßwald, Evelyn Fischer, Christoph Degenhart, Marcus Quinkler, Martin Bidlingmaier, Anna Pallauf, Katharina Lang, Thomas Mussack, Klaus Hallfeldt, Felix Beuschlein, and Martin Reincke

Objective

Adrenal vein sampling (AVS) is a technically demanding procedure required for the identification of suitable candidates for unilateral adrenalectomy in primary aldosteronism. Recently, somatic KCNJ5 K+-channel mutations in aldosterone-producing adenoma (APA) patients have been shown to influence steroid gradients during AVS. These and other recently identified genetic modifiers (ATP1A1 and ATP2B3) might affect the final diagnosis and treatment of the affected patients.

Design

Fifty-nine patients with APAs who had undergone successful AVS (adrenal vein cortisol:peripheral cortisol ratio ≥2) and had undergone a mutation analysis of their tumor tissue were studied. The mutation status of the APAs was as follows: 19 KCNJ5 mutations, eight ATPase mutations (five ATP1A1 and three ATP2B3), and 32 patients with none of these mutations.

Methods

The lateralization index (ratio of aldosterone:cortisol on the side of the adenoma to aldosterone to cortisol on the contralateral side) and the contralateral suppression index (ratio of aldosterone:cortisol on the contralateral side to aldosterone to cortisol in the periphery) were calculated for the KCNJ5-mutated, ATPase-mutated, and the KCNJ5/ATPase mutation-negative APA patients.

Results

The lateralization indices of the ATPase mutation carriers had a median of 19.9 compared with a median of 16.0 in the KCNJ5 mutation carriers and that of 20.5 in the KCNJ5/ATPase mutation-negative patients. The contralateral suppression indices of the ATPase-mutated patients had a median of 0.1 compared with a median of 0.4 in the KCNJ5 mutation carriers and that of 0.2 in the KCNJ5/ATPase mutation-negative patients. The differences between the genetic groups were not statistically significant.

Conclusions

We did not find evidence for a clinically important impact of mutation status on steroid gradients during AVS.

Free access

ACTH after 15 min distinguishes between Cushing's disease and ectopic Cushing's syndrome: a proposal for a short and simple CRH test

Katrin Ritzel, Felix Beuschlein, Christina Berr, Andrea Osswald, Nicole Reisch, Martin Bidlingmaier, Harald Schneider, Jürgen Honegger, Lucas L. Geyer, Jochen Schopohl, and Martin Reincke

Objective

The aim of the present study was to validate criteria of corticotropin-releasing hormone (CRH) stimulation and 8 mg dexamethasone suppression (high-dose dexamethasone suppression, HDDS) to distinguish the etiology of ACTH-dependent Cushing's syndrome.

Subjects and methods

We retrospectively analyzed cortisol and ACTH after the injection of 100 μg human CRH in confirmed Cushing's disease (CD, n=78) and confirmed ectopic Cushing's syndrome (ECS, n=18). Cortisol and ACTH increase (in percentage above basal (%B)) at each time point, maximal increase (Δmax %B), and area under the curve (AUC %B) were analyzed using receiver operator characteristics (ROC) curve analyses. Cortisol suppression (%B) after 8 mg of dexamethasone was evaluated as a supplementary criterion.

Results

An increase in ACTH of ≥43%B at 15 min after CRH was the strongest predictor of CD, with a positive likelihood ratio of 14.0, a sensitivity of 83%, a specificity of 94%, a positive predictive value of 98% and a negative predictive value of 58%. All of the other criteria of stimulated ACTH and cortisol levels were not superior in predicting CD in response to CRH injection. The addition of cortisol suppression by dexamethasone did not increase the discriminatory power. However, the combination of a positive ACTH response at 15 min and a positive HDDS test excluded ECS in all cases.

Conclusion

The present findings support the use of plasma ACTH levels 15 min after the injection of human CRH as a response criterion for distinguishing between CD and ECS. The addition of the HDDS test is helpful for excluding ECS when both tests are positive.

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Pharmacokinetics and pharmacodynamics of GH: dependence on route and dosage of administration

Alexandra Keller, Zida Wu, Juergen Kratzsch, Eberhard Keller, Werner F Blum, Astrid Kniess, Rainer Preiss, Jens Teichert, Christian J Strasburger, and Martin Bidlingmaier

Objective: Pharmacokinetic and pharmacodynamic data after recombinant human GH (rhGH) administration in adults are scarce, but necessary to optimize replacement therapy and to detect doping. We examined pharmacokinetics, pharmacodynamics, and 20 kDa GH after injection of rhGH at different doses and routes of administration.

Design: Open-label crossover study with single boluses of rhGH.

Methods: Healthy trained subjects (10 males, 10 females) received bolus injections of rhGH on three occasions: 0.033 mg/kg s.c., 0.083 mg/kg s.c., and 0.033 mg/kg i.m. Concentrations of 22 and 20 kDa GH, IGF-I, and IGF-binding proteins (IGFBP)-3 were measured repeatedly before and up to 36 h after injection.

Results: Serum GH maximal concentration (C max) and area under the time-concentration curve (AUC) were higher after i.m. than s.c. administration of 0.033 mg/kg (C max 35.5 and 12.0 μ g/l; AUC 196.2 and 123.8). C max and AUC were higher in males than in females (P < 0.01) and pharmacodynamic changes were more pronounced. IGFBP-3 concentrations showed no dose dependency. In response to rhGH administration, 20 kDa GH decreased in females and remained suppressed for 14–18 h (low dose) and 30 h (high dose). In males, 20 kDa GH was undetectable at baseline and throughout the study.

Conclusions: After rhGH administration, pharmacokinetic parameters are mainly influenced by route of administration, whereas pharmacodynamic variables and 20 kDa GH concentrations are determined mainly by gender. These differences need to be considered for therapeutic use and for detection of rhGH doping.