A quantitative LC-MS/MS method for insulin-like growth factor 1 in human plasma.

BACKGROUND: Insulin-like growth factor 1 (IGF1) is a biomarker with various applications in medicine and also in doping control. METHODS: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed that employs 15N-IGF1 as an internal standard. The method features urea-based IGF1/IGFBP-complex dissociation which is directly followed by tryptic digestion. Following solid-phase extraction (SPE) sample clean-up of the digest, IGF1 is detected by means of two signature peptides that enable quantification of total IGF1 as well as discrimination between IGF1 proteoforms with 'native' and modified or extended N-terminal sequences. RESULTS: Our method is capable of measuring plasma IGF1 concentrations over the clinically relevant range of 10-1000 ng/mL and was validated according to regulatory guidelines. Comparison with the IDS-iSYS IGF1 immunoassay revealed good correlation (R2>0.97) and no proportional bias between both assays was observed after normalizing the results against the WHO reference standard for IGF1 (02/254). Evaluation of several commercially available IGF1 preparations showed varying responses which were due to inconsistencies in purity and absolute amount of IGF1 present in these products. CONCLUSIONS: Our LC-MS/MS method introduces urea-based dissociation of IGF1/IGFBP-complexes to enable reliable quantification of IGF1 in plasma. Furthermore, the method is able to detect clinically relevant IGF1 levels without an enrichment procedure at the protein-level and thereby minimizes the risk of losing IGF1 proteoforms during sample preparation.

Disagreement in normative IGF-I levels may lead to different clinical interpretations and GH dose adjustments in GH deficiency.

INTRODUCTION AND BACKGROUND: Normative data for the iSYS IGF-I assay have been published both in the VARIETE cohort and by Bidlingmaier et al. OBJECTIVE: To investigate whether normative data of the VARIETE cohort lead to differences in Z-scores for total IGF-I and clinical interpretation compared to normative data of Bidlingmaier et al. DESIGN: We used total IGF-I values previously measured by the IDS-iSYS assay in 102 GH-deficient subjects before starting GH treatment and after 12 months of GH treatment. Z-scores were calculated for all samples by using the normative data of the VARIETE cohort and by the normative data reported by Bidlingmaier et al. RESULT: Before GH treatment, Z-scores calculated by using the normative data of the VARIETE cohort were significantly lower than those calculated by the normative data of Bidlingmaier et al: -2.40 (-4.52 to +1.31) (mean [range]) vs. -1.41 (-3.14 to +1.76); P < .001). After 12 months of GH treatment, again the Z-scores based on the normative data of the VARIETE cohort were significantly lower than those based on the normative data of Bidlingmaier et al: -0.65 (-4.32 to +2.79) vs 0.21 (-3.00 to +3.28); P < .001). CONCLUSION: IGF-I Z-scores in 102 GH-deficient subjects differed significantly when normative data from two different sources were used. In daily clinical practice, this would most likely have led to different clinical interpretations and GH dose adjustments.

Interlaboratory agreement of insulin-like growth factor 1 concentrations measured by mass spectrometry.

BACKGROUND: Insulin-like growth factor 1 (IGF-1)(7) is a key mediator of growth hormone (GH) action and a well-characterized biomarker of GH abuse. Current immunoassays for IGF-1 suffer from poor concordance between platforms, which makes comparison of results between laboratories difficult. Although previous work has demonstrated good interlaboratory imprecision of LC-MS/MS methods when plasma is supplemented with purified proteins, the interlaboratory imprecision of an endogenous protein in the nanogram-per-milliliter concentration range has not been reported. METHODS: We deployed an LC-MS/MS method to quantify serum IGF-1 in 5 laboratories using 5 different instruments and analyzed 130 healthy human samples and 22 samples from patients with acromegaly. We determined measurement imprecision (CV) for differences due to instrumentation, calibration curve construction, method of calibration, and reference material. RESULTS: Instrument-dependent variation, exclusive of digestion, across 5 different instrument platforms was determined to be 5.6%. Interlaboratory variation was strongly dependent on calibration. Calibration materials from a single laboratory resulted in less variation than materials made in individual laboratories (CV 5.2% vs 12.8%, respectively). The mean imprecision for 152 samples between the 5 laboratories was 16.0% when a calibration curve was made in each laboratory and 11.1% when a single-point calibration approach was used. CONCLUSIONS: The interlaboratory imprecision of serum IGF-1 concentrations is acceptable for use of the assay in antidoping laboratories and in standardizing results across clinical laboratories. The primary source of variability is not derived from the sample preparation but from the method of calibration.

The current status of IGF-I assays--a 2009 update.

For almost three decades, the measurement of circulating IGF-I has constituted a highly important biochemical tool in the management of GH disorders. In fact, in acromegaly the importance of circulating IGF-I has increased following the introduction of the GH receptor antagonist pegvisomant, as the use of this drug makes it impossible to use circulating GH as a monitor of disease activity. In addition, determination of circulating IGF-I constitutes a valuable scientific tool in various research areas, from epidemiological investigations through clinical trials and experimental studies. The multiple facets of IGF-I physiology and patho-physiology may explain why numerous endocrine laboratories have invested in IGF-I assays, by means of either in-house assays or commercial kits. However, despite its widespread use, the measurement of IGF-I is by no means trivial. On the contrary, the pronounced binding of IGF-I to the high-affinity IGF-binding proteins (IGFBPs) constitutes a notorious source of error, which has necessitated the development of methods that more or less successfully circumvent interference from the IGFBPs. Furthermore, there are some unsolved issues with the international standardization of the different IGF-I assays and there is no consensus regarding the procedures used when collecting and storing samples for measurement of circulating IGF-I. The aim of this review is to discuss the current state of the art of IGF-I immunoassays and to present the current analytical problems with IGF-I measurements. Finally, we would like to suggest an agenda that may be used when trying to produce internationally accepted uniform requirements for future IGF-I assays.

Establishment of reference values for endocrine tests. Part VII: growth hormone deficiency.

BACKGROUND: Plasma insulin-like growth factor (IGF-I) concentration can be used as a rough indicator of the growth-hormone status. However, for the diagnosis of growth hormone deficiency, dynamic tests are required. The growth hormone (GH) response in the insulin tolerance test (ITT) is considered to be the gold standard in this respect. An alternative for the ITT is the GHRH/ GHRP-6 test, which has fewer side effects. In this study we established reference values for IGF-I levels and for the GH response in both dynamic tests. METHODS: We studied 296 subjects recruited from the general population, equally distributed according to sex and aged between 20 and 70 years. Serum IGF-I level was measured in all subjects and an insulin tolerance test (0.15 U/kg Actrapid iv) and GHRH/GHRP-6 test (1 microg GHRH/kg and 1 microg GHRP-6/kg) were performed in 49 subjects. RESULTS: In multivariate analyses both IGF-I and the GH response in the ITT were significantly influenced by age, whereas the GH response in the GHRH/GHRP-6 test was significantly affected by BMI. There was no sex difference in IGF-I and in the GHRH/GHRP-6 test, but in the ITT males had a higher GH peak. There was a significant correlation between the GH responses in both tests, and the GH response was significantly higher in the GHRH/GHRP-6 test than in the ITT. Age-adjusted reference values were established for each test. CONCLUSION: We have established age-adjusted reference values for serum IGF-I and for the GH response in the ITT and GHRH/GHRP-6 test.

The First International Standard For Insulin-like Growth Factor-1 (IGF-1) for immunoassay: preparation and calibration in an international collaborative study.

The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) has recognized the need for an International Standard for Insulin-like Growth Factor-1 (IGF-1) for the calibration of immunoassays and for the monitoring of the content of therapeutic products. The objective of the study reported here was the characterization of a candidate standard for IGF-1 in an international collaborative study carried out by 18 laboratories in nine countries, by comparison with (i) a primary calibrant characterized by amino acid analysis and UV spectroscopy, and (ii) the existing International Reference Reagent coded 87/518 by HPLC, immunoassay and bioassay. The study was designed as follows: Phase I involved the establishment of a primary calibrant of rhIGF-1, containing approximately 1.0mg rhIGF-1 per vial. A defined value was assigned to the primary calibrant by amino acid analysis (AAA) and UV spectroscopy. Phase II involved calibration of the candidate standard in terms of the primary calibrant by HPLC, with confirmatory data from immunoassay and bioassay. Results from Phase I confirmed the primary calibrant as containing 1.045 mg per vial. Although there was some variability among laboratory estimates of IGF-1 in the proposed standard using the different methods in Phase II, the estimates by the various methods were in broad agreement. On the basis of the results reported here, the World Health Organization (WHO) has established the preparation coded 02/254 as the First International Standard for Insulin-like Growth Factor-1, human, recombinant, for immunoassay with an assigned content of 8.50 microg per ampoule. Details of how to order the standard can be found at www.nibsc.ac.uk.

A simple and cost-effective approach to assessment of pituitary adrenocorticotropin and growth hormone reserve: combined use of the overnight metyrapone test and insulin-like growth factor-I standard deviation scores.

CONTEXT: The insulin tolerance test (ITT) is the gold standard for assessment of ACTH and GH reserve in patients with suspected hypopituitarism. It is labor intensive and costly. OBJECTIVE: The objective of the study was to determine whether use of the overnight metyrapone test (OMT) and plasma IGF-I sd scores (SDS) could provide a cost-effective alternative to the ITT. DESIGN: This was a retrospective chart review. SETTING: The study was conducted at a teaching hospital. PARTICIPANTS AND INTERVENTION: Charts from 100 patients with organic pituitary disorders were reviewed. All underwent the OMT unless 0900 h plasma cortisol was less than 80 or greater than 450 nmol/liter when ACTH deficiency or ACTH sufficiency, respectively, was diagnosed. Patients were considered GH deficient if the age-related IGF-I SDS was less than -3 or if they had three or more other pituitary hormone deficiencies. Patients were considered GH sufficient if age-related IGF-I SDS was greater than the 95th centile established from patients with known GH deficiency. Thirty-three underwent an ITT. MAIN OUTCOME MEASURES: The proportion of patients in whom ACTH and GH reserve could be assessed using OMT/IGF-I SDS was measured. The concordance with results was obtained from ITT. RESULTS: Fifty-five patients were ACTH sufficient and 45 were ACTH deficient. Twenty-one were GH sufficient and 33 were GH deficient based on IGF-I SDS and other pituitary hormone deficiencies, whereas 46 could not be classified. There was near-uniform concordance between OMT/IGF-I SDS and ITT. Initial investigation using OMT/IGF-I SDS resulted in a significant cost saving. CONCLUSIONS: ACTH and GH reserve can be accurately and cost-effectively investigated using OMT/IGF-I SDS in approximately 50% of patients with organic pituitary disorders.

Normal levels of serum IGF-I: determinants and validity of current reference ranges.

Insulin like growth factor I (IGF-I) represents the key marker for the evaluation of Growth hormone (GH) status. As a large number of determinants including age, gender, genetic factors, nutrition, and disease states influence IGF-I serum levels, accurate normative data are essential to translate patient data into diagnostic meaning or even use IGF-I levels for adequate monitoring of patients with an over- or under-active GH axis. Even though reference ranges have been developed in large cohorts of healthy subjects, the dependency of these data on a given assay technology argues for assay specific normative data for IGF-I.

Serum IGF-I levels in the diagnosis and monitoring of acromegaly.

Insulin-Like Growth Factor-I (IGF-I) is a reliable marker of disease activity and growth hormone (GH) status in acromegaly, but its clinical utility has been hampered over the years by various issues including a lack of robust reference range data and variability in assay sensitivity and specificity. In acromegaly IGF-I correlates well with GH activity and nadir GH on oral glucose tolerance test (OGTT) and is the most sensitive and specific test in diagnosis, where serum IGF-I is persistently seen to be elevated to a range that is distinct from that in healthy individuals. However it should not be relied on exclusively for diagnosis or used as the sole indication of disease severity and GH burden. Successful medical or surgical treatment of acromegaly is usually associated with normalisation of serum IGF-I but there is discordance between GH and IGF-I in some patients. Patients with a normal IGF-I but an abnormal GH suppression to OGTT are at risk of relapse and therefore it should not be used alone to establish disease remission. In contrast to the diagnosis of acromegaly, there is also considerable overlap in serum IGF-I with normality after primary treatment of disease, even in the presence of persisting GH excess. Gender, age and prior radiotherapy alters the relationship between GH and IGF-I and reliance on one marker of disease activity such as IGF-I is particularly precarious in certain disease states. However an elevated serum IGF-I has been shown to be associated with excess mortality and normalising IGF-I normalises mortality making it a useful marker. The tightening up of the assays means that establishing absolute concentrations as well as standard deviation scores are essential to allow cross-study comparisons. This becomes especially important in the use of Pegvisomant, where IGF-I becomes the sole biochemical marker of disease activity.

Utility of free IGF-I measurements.

For nearly 30 years, the endogenous bioactivity of insulin-like growth factor I (IGF-I) has been estimated by its circulating concentrations of immunoreactive IGF-I, obtained after either removal or inactivation of the IGF-binding proteins (IGFBPs), and today serum/plasma total IGF-I serves as a useful parameter in the diagnosis and clinical control of growth hormone (GH) disorders. Different assays for the measurement of free, unbound IGF-I were introduced more than a decade ago. Nevertheless, this measurement remains controversial, and in daily clinical practice serum total IGF-I has retained its position as the most widely used IGF-related measurement in GH disorders. This review will provide a survey of data on free versus total IGF-I, with particular reference to GH disorders. As it will be clear, there is reasonable clinical evidence to conclude that both in the diagnosis of as well as during treatment of patients with GH disorders, serum/plasma total IGF-I should remain the primary IGF-related measurement. However, in certain patients the inclusion of free IGF-I may be useful and therefore, some guidelines for the inclusion of free IGF-I measurements will be given.

Defining normalcy of the somatotropic axis: an attainable goal?

The diagnoses of acromegaly and dwarfism require biochemical confirmation of abnormal GH and IGF-1 concentrations. The same parameters are used for therapeutic decisions, i.e. initiation or termination of particular treatments. Therefore, reliable and epidemiologically and statistically proven criteria of normalcy for GH and IGF-1 are required for these tasks to be accomplished. Despite major progress in all these areas, the definition of what constitutes "normalcy" of the somatotropic axis is still lacking. Using an example of acromegaly, we discuss the contradictions and the uncertainties of the biochemical diagnosis of this disease.

IGF-I measurements in the monitoring of GH therapy.

Growth hormone replacement therapy has been used regularly in adult Growth hormone deficiency since the availability of recombinant GH in the 1980's. GH replacement improves quality of life, bone turnover markers, cardiovascular risk markers and adverse body composition. Originally, GH doses in replacement regimes were determined by weight and surface area and dose increases based on body composition outcomes analogous to pediatric practice. These regimens led to significant side effects related to excess GH, arthralgias, headaches and peripheral edema and IGF-I levels above the upper limit of the reference range. Newer treatment regimes therefore account for known factors affecting serum GH and IGF-I levels, i.e. age, gender, estrogen replacement and pre-treatment IGF-I levels. Monitoring is now via clinical symptomatology combined with serum total IGF-I levels, potentially this avoids excessive GH exposure and allows monitoring of compliance and dose titration. There is a lack of data relating IGF-I to biological endpoints, but analysis suggests that dose titration of IGF-I to the upper half of the age and gender related reference range is acceptable. The use of reliable IGF-I assays and extensive age and gender related reference ranges is necessary and centralized monitoring is preferable. Free IGF-I and bioavailable IGF-I measurements are available but their use in the monitoring of GH replacement remains to be determined.

Applicability of recently established reference values for serum insulin-like growth factor 1: A comparison of two assays--an (automated) chemiluminescence immunoassay and an enzyme-linked immunosorbent assay.

Analysis and interpretation of insulin-like growth factor 1 in serum (IGF-1) is a principal diagnostic and follow-up tool in growth hormone-related disorders and is becoming of interest for many other disorders. Only for the automated chemiluminescence immunoassay Nichols Advantage have age- and sex-specific reference values based on a large population and valid for different laboratories been established. The aim of the present study was to compare two different assays (the automated chemiluminescence immunoassay and the enzyme-linked immunosorbent assay DSL-10-2800) in order to prove applicability of recently published reference values. The study included 95 serum samples from 88 patients, adults as well as children, with different or no endocrine disorders, and acromegalic as well as growth hormone-deficient patients. IGF-1 measurements were performed with both methods. The results have shown a very high correlation between the IGF-1 values obtained with the two assays (r=0.971, p<0.0001). In conclusion, the reference values established for the chemiluminescence assay are applicable also for the enzyme-linked immunosorbent assay.

Clinical efficacy of insulin-like growth factor-1 in a patient with autoantibodies to insulin receptors: a case report.

The type B insulin-resistance syndrome is characterized by the presence of anti-insulin receptor antibodies that cause severe insulin resistance. Treatments including steroids, cyclophosphamide, plasmapheresis, or insulin-like growth factor-1 (IGF-1) are chosen according to severity of insulin resistance. We describe a patient with type B insulin resistance syndrome who was treated successfully with human recombinant (hr) IGF-1, although this treatment provoked a severe allergic reaction. An elderly man with impaired glucose tolerance and unpredictable hypoglycemic episodes which were gradually worsening increased in hemoglobin (Hb)A1c concentration from 6.5 to 13.4%. His fasting and postprandial hyperglycemia were associated with severe hyperinsulinemia. The patient was diagnosed with type B insulin-resistance syndrome by the presence of anti-insulin receptor antibodies. Double-filtration plasmapheresis, plasma exchange, and immunosuppressive therapy with cyclophosphamide and cyclosporin all failed to suppress anti-insulin receptor antibodies more than transiently. When we attempted the treatment by daily administration of hrIGF-1, fasting and postprandial plasma glucose concentrations became normal and HbA1c levels decreased to 7.1% over 2 months, until on one occasion administration resulted in anaphylaxis. After the patient became stable, desensitization therapy was performed successfully, and hrIGF-1 could be administered again with the plasma glucose returning. We concluded that IGF-1 therapy was an effective treatment choice for type B insulin-resistance syndrome in cases whose plasma exchange and immunosuppressive therapy have failed.

How much insulin-like growth factor-I (IGF-I) circulates?: impact of standardization on IGF-I assay accuracy.

Insulin-like growth factor-I (IGF-I, somatomedin-C) is a peptide hormone which plays an important role in growth regulation. Accurate measurements of circulating IGF-I levels in plasma samples are an important part of many studies on growth and development, and therefore many assays have been developed for this purpose. We have found that assay standardization has a major impact on IGF-I quantification. Most IGF-I assays are calibrated against the World Health Organization (WHO) International Reference Reagent (IRR) for IGF-I Immunoassays (87/518). The protein content assigned to WHO IRR 87/518 was a consensus value from a multicentre WHO collaborative study. Here we present physico-chemical data showing that WHO IRR 87/518 is Met(-1) IGF-I of low purity (44%), and that the assigned protein content is somewhat higher than the <> value determined by quantitative amino acid analysis. We show that assays calibrated against WHO IRR 87/518 report total IGF-I concentrations that are in excess of actual values by approximately two-fold. For this reason much of the IGF-I concentration data in the literature, which are based on assays calibrated against WHO IRR 87/518, are of questionable accuracy.

Investigaciones Experimentales sobre el efecto del factor de crecimiento semejante a la insulina tipo 1 en el síndrome de intestino corto

Luego de la resección intestinal masiva el intestino remanente experimenta cambios mucosos muy importantes para compensar la remoción de la superficie absortiva. Estos cambios pueden ser disminuidos aportando nutrientes enterales y factores del crecimiento exógenos tales como el IGF-1. La adaptación colónica puede ser aumentada mediante la adición de una mejora en la absorción del intestino delgado. Existe evidencia experimental preliminar de que el aporte intraluminar de IGF-1 produce efectos tróficos directos sobre el intestino delgado, probablemente mediados por mecaniamos paracrinos.

The International Reference Reagent for insulin-like growth factor-I.

Three preparations of recombinant DNA-derived insulin-like growth factor-I (IGF-I) were obtained, prepared in ampoules coded 86/522, 86/720 or 87/518, and evaluated as candidate International Reference Reagents in an international collaborative study (nine laboratories in four countries) in response to a request by the World Health Organization (WHO). Immunoassay dose-response curves for each of the three preparations did not in general differ significantly from those of local standards or from those of ampouled preparations of serum-derived IGF-I which were included in the study. The estimates of ampoule contents in terms of local standards showed considerable heterogeneity; the between-laboratory variability of estimates in terms of local standards was ten times greater than the inherent variability of estimates from these systems as estimated from comparisons of coded duplicates. Bioassay data were limited, and those available were inconsistent with immunoassay data. Of the three preparations, ampoules coded 86/720 were derived from an IGF-I preparation that was heterogeneous by high-performance liquid chromatography, and stability data for the preparation 86/522 were anomalous. As a result, the ampouled preparation coded 87/518 has been established by WHO as the International Reference Reagent for IGF-I for immunoassay, with an assigned ampoule content of 3.1 micrograms/ampoule, and is available from the National Institute for Biological Standards and Control.