Determination of IGFs and their binding proteins

Best Practice & Research Clinical Endocrinology & Metabolism 27 (2013) 771–781

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Determination of IGFs and their binding proteins Rikke Hjortebjerg, MSc, Research Assistant a, Jan Frystyk, MD, PhD, DMSc, Professor a, b, * a

Medical Research Laboratory, Department of Clinical Medicine, Faculty of Health, Aarhus University, DK-8000 Aarhus C, Denmark b Department of Endocrinology and Internal Medicine, Aarhus University Hospital, DK-8000 Aarhus C, Denmark

Keywords: IGF-I IGFBP immunoassay assay development pre-analytical post-analytical consensus statement

The worldwide clinical and scientific interest in peptides belonging to the insulin-like growth factor (IGF) system has brought along a call for standardization of assays used to quantify the different IGF related proteins. This relates in particular to the measurement of IGF-I, which has stood the test of time as an important biochemical tool in the diagnosis and treatment of growth hormone (GH) related disorders. The first international consensus statement on the measurement of IGF-I in 2011 represents an important milestone and will undoubtedly improve commutability of reference ranges for IGF-I and clinically applicable cut-off values. By contrast, there is no consensus addressing the measurements of the other IGF-related peptides. Nevertheless, measurement of these peptides may be of interest, either as additional tools in GH disorders or as prognostic biomarkers of various diseases. Therefore, standardization of assays for the other IGF-related peptides is highly relevant. This chapter discusses the recent consensus on IGF-I measurements and how this approach may be applied to measurement of the other IGF-related peptides. In addition, assay pitfalls, pre- and post-analytical challenges, alternative methods for IGF-I measurements and potential assays of tomorrow will be discussed. Ó 2013 Elsevier Ltd. All rights reserved.

* Corresponding author. Medical Research Laboratory, Aarhus University Hospital, Nørrebrogade 44, DK-8000 Aarhus C, Denmark. Tel.: þ45 7846 2166; Fax: þ45 7846 2150. E-mail address: [email protected] (J. Frystyk). 1521-690X/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.beem.2013.08.010

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Introduction to the insulin-like growth factor system The established components of the IGF system include IGF-I and the structurally related IGF-II, six high-affinity IGF-binding proteins (IGFBPs; IGFBP-1 to -6) and the acid labile unit (ALS). Measurement of serum IGF-I levels has stood the test of time as an important tool in the diagnosis and treatment of growth hormone (GH) related disorders [1], making IGF-I by far the most clinically relevant peptide within the IGF system. This may explain why the consensus does not address the measurement of other IGF-related proteins [2]. Nevertheless, it appears that many of the other IGF-related peptides may also have some clinical relevance, either as additional tools in GH disorders (IGFBP-3 and ALS) [1], or as prognostic biomarkers of various diseases, for instance cancer (IGFBP-2) [3], type 2 diabetes (IGFBP-1) [4] and atherosclerosis (IGFBP-4 fragments) [5]. Therefore, standardization of assays for the other IGFrelated peptides is highly relevant. The IGFBPs bind close to 99% of the circulating IGFs with high affinity, hereby affecting the half-life of the circulating IGF-pool as well as its tissue accessibility [6,7]. Although the IGFBPs usually inhibit the IGFs from activating their receptors in vitro, numerous in vivo studies have demonstrated that the IGFBPs may also potentiate IGF-mediated actions. For this reason the IGFBPs are often referred to as modulators of IGF-action [8–11]. Additionally, in vitro experiments have demonstrated that the IGFBPs possess biological effects independent of the IGFs [12]. To further complicate the system, a number of IGFBP-specific proteases partake in the regulation of IGF-action. Following enzymatic cleavage the ligand affinity of the IGFBPs becomes markedly reduced, leading to dissociation of the IGFs, which hereby becomes accessible for receptor activation. Thus, enzymatic cleavage of the IGFBPs is considered to play a key role in controlling IGF-action [10,12]. IGF-I and IGF-II serve as ligands for the ubiquitously expressed, cell surface-associated IGF-I receptor (IGF-IR), albeit with different affinities [13,14]. Key downstream pathways following IGF-IR activation include the phosphatidylinositol 3-kinase (PI3K) pathway, which primarily favours metabolic, insulin-mimicking effects and the mitogen-activated protein kinase (MAPK) pathway, which primarily favours mitogenesis. Furthermore, IGF-IR activation prevents apoptosis. For further details the reader is referred to excellent reviews [15–17]. IGF-II also interacts with the insulin-like growth factor/mannose-6-phosphate receptor (IGF-IIR). This receptor is structurally and functionally distinct from the IGF-IR as it contains no intrinsic tyrosine kinase activity. One of the main functions of the IGF-IIR is to serve as an IGF-II scavenger, clearing and degrading IGF-II from the extracellular environment without activating intracellular signalling cascades. Thus, many, but not all, IGF-II actions may be explained by its interaction with the IGF-IR [18,19], and consequently, the IGF-IR may be considered as the primary target for both growth factors. For further information on IGF-II please see [20–23]. The clinical interest in IGF-I originates from its intimate association with GH, which is by far the most important regulator of IGF-I. Positive correlations between the integrated 24-h secretion of GH and serum IGF-I have been demonstrated in healthy prepubertal and pubertal children [24], healthy adults [25], GH deficient patients [25] as well as acromegalic patients before and after treatment [26]. These findings constitute the scientific rationale for using serum IGF-I as a marker of the spontaneous GH secretion as well as a marker of treatment, whether this involves replacement therapy with recombinant human GH or treatment with GH antagonists such as pegvisomant and somatostatin analogues. However, it is important to stress that only in selected patient cases serum IGF-I can serve as a standalone test in the diagnosis of GH deficiency [27], whereas serum IGF-I is performing better in the diagnosis of GH excess [1,28]. The relationship between IGF-I and GH is bidirectional, as IGF-I inhibits GH secretion at the level of hypothalamus and pituitary [29]. This bidirectional relationship has been clearly demonstrated in humans following administration of exogenous IGF-I, which leads to blunting of the GH secretion [30]. Insulin is the second most important regulator of IGF-I and stimulates IGF-I action via two different mechanisms. When the liver becomes exposed to insulin delivered via the portal vein, the hepatic synthesis of IGFBP-1 is rapidly suppressed. IGFBP-1 down regulates free and bioactive IGF-I in vitro and most likely this impacts IGF-I action in vivo [11]. Secondly, insulin stimulates the hepatic GH receptor density and consequently the hepatic sensitivity to GH [31]. The latter may explain why obese subjects, who are likely to be insulin resistant and hyperinsulinemic, are more sensitive to GH than lean subjects

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[32]. Thus, portal vein insulin serves to increase IGF-I action via down regulation of IGFBP-1 and by increasing the hepatic sensitivity to GH and hence the production of IGF-I [33]. IGFBP-3 is the most abundant IGFBP and binds as much as 75% of the circulating IGF-pool in longlived 150 kilo Dalton (kDa) ternary complexes with ALS. Due to their molecular size, the ternary complexes are essentially confined to the circulation [12]. IGFBP-3 and ALS are both regulated by GH, but in adults neither of these proteins has proven to be superior to IGF-I in the diagnosis and treatment of GH disorders and therefore they have not gained any major role in adult endocrine practice. In young children, however, IGFBP-3 may have a greater discriminatory value than IGF-I [1,34]. The reason that IGFBP-3 and ALS are generally less useful as predictors of GH status is related to the fact that about twothirds of the ternary complexes contain IGF-II, which is not regulated by GH. IGFBP-2 is the second most abundant IGFBP and like IGFBP-1, it links the IGF system with insulin sensitivity, glucose metabolism and in addition, leptin and cancer [8,35–37]. However, our knowledge about the regulation and function of IGFBP-2 is incomplete. The same is true for IGFBP-4 to -6 and as it is beyond the scope of this article to describe these IGFBPs in further detail, the reader is referred to recent reviews [10,38–42]. Structural relationship between IGFS and IGFBPS IGF-I and -II were chemically characterized and structurally delineated in 1978. The homologous peptides share 67% sequence similarity and display high sequence similarity (w50%) to the A and B domains of insulin. However, both IGFs contain additional and unique C and D domains [43]. The six high-affinity IGFBPs comprise a family of well-defined and structurally related proteins containing 216–289 amino acids [44]. Although the IGFBPs differ in primary structures, they share distinctive structural and functional characteristics and all of them bind IGF-I or IGF-II on a 1:1 M basis. The IGFBPs all share a highly conserved protein structure separated into three distinct domains of approximately equal size, with the conserved amino (N)- and carboxy (C)-terminal domains connected by a ‘linker’ (L)-domain that shows little similarity among the IGFBPs [12]. The specific IGF-binding affinities differ between the N- and C-terminal domains. The N-terminal domain has been demonstrated to bind IGF with a 10–1000 times lower affinity than do full-length IGFBPs. Likewise, the affinity of the C-terminal domain is also greatly reduced when it is no longer part of an intact IGFBP [45,46]. By contrast, the L-domain shows no direct involvement in IGF-binding. Its contributions to IGF-binding is most likely related to its ability to promote a well organised tertiary structure. Interestingly, posttranslational modifications have been found only in the L-domain. Among these modifications are phosphorylations, glycosylations, proteolysis and adherence of the IGFBP to cell surfaces [12]. Some of the alterations do not appear to affect IGF-binding, whereas others change IGFBP stability, half-life, binding-affinity etc. For instance, phosphorylation of IGFBP-1 increases the affinity for IGF-I by approximately six-fold, whereas adherence to the cell surface lowers IGFBP affinity and potentiates IGF actions [47,48]. Determination of IGFs and IGFBPs Immunoassays constitute the mainstay and the technique of choice for measuring circulating IGFs and IGFBPs and they are ubiquitously applied in clinical medicine as well as in research. This chapter provides an overview of conventional immunoassays and alternative methods for measuring IGFs and IGFBPs. In addition, we discuss the analytical difficulties in determining IGF and IGFBP concentrations. First, however, we will bring pre-analytical and post-analytical challenges to the attention of the reader. Pre- and post-analytic challenges It is important to realize that it takes more than a high quality immunoassay to yield reliable and reproducible results. According to recent reviews, improvements in reliability and standardization of analytical techniques, reagents and instrumentation have resulted in a 10-fold reduction in the analytical error rate, hereby improving medical decision-making and patient care. As a result, the

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majority of errors now occur outside the analytical phase, i.e. pre- and post-analytically [49,50]. This call for careful handling becomes evident when reviewing the existing literature describing pitfalls in sample handling procedures from patient to IGF laboratory. Although limited in number, these studies clearly show that the measured concentrations of IGF-I and IGFBP-3 may be influenced by preanalytical procedures. Time to centrifugation and separation of blood cells appears to have a major impact, but sample type, i.e., serum or plasma, has also been reported to affect the reported levels of IGFs, IGFBPs and ALS [51,52]. Indeed, these issues have been mentioned in the recent consensus statement on the measurement of IGF-I, which recommends the use of serum for measurement of IGF-I and further that blood is processed within two hours after collection to avoid artificial increases in the results [2]. Shipment of samples to the laboratory is also a challenge in terms of temperature exposure and duration of the transport. In the ideal world, standard operational procedures for all steps in the pre-analytical handling should be developed, tested and published for every IGF-related immunoassay (in particular commercially available methods), but we acknowledge that these goals are hard to reach. Thus, we recommend that at the very least, that each IGF-laboratory tests the pre-analytical procedures for sample processing. Another issue that needs to be considered is under which circumstances blood samples have to be collected. Drawing of blood for GH measurements requires highly standardized conditions, whereas serum for measurement of IGF-I and IGF-II may be collected during the day, without taking meals into consideration [51]. For the other IGF related proteins, data indicate some effect of meals or oral glucose on the levels of IGFBP-2, -3 and -4 [51,53,54] and a marked effect on IGFBP-1 [55]. Thus, if IGFBPs are to be measured, sample collection should be standardized. When it comes to the post-analytical phase in IGF and IGFBP measurements, the biggest challenge relates to the availability of an appropriately sized reference material, allowing for age-specific reference values. This necessity is based on the fact that all IGF related peptides but IGFBP-4 exhibit age dependent variation [42,52,56]. This constitutes a particular clinical problem in regards to IGF-I, which in addition, for all ages are characterized by large inter-individual variation and a strong association with the pubertal GH surge, which varies in a gender specific manner. Therefore, to be on safe grounds when interpreting an IGF-I value, one has to compare it against age-adjusted reference values in adults, and against gender- and age-adjusted values in children between 6 and 18 years [2,51]. The importance of an appropriately sized reference material has been elucidated by Massart and Poirier, who showed that in acromegalic patients, the number of patients categorized as sufficiently treated on the basis of an IGF-I serum determination was highly dependent on the size of the reference material [57]. Immunoassays for determination of IGFs The first determinations of IGF-I were based on bioassays and later by competitive membrane binding-assays [11,58]. The general lack of specificity and precision in these early assays was overcome in 1977 by the development of the first radioimmunoassay (RIA) for determination of IGF-I [59]. Today, numerous non-radioactive commercial and in-house IGF-I assays have been developed, of which the enzyme-linked immunosorbent assay (ELISA) can be considered the most successful. Alternative assay formats that further improve sensitivity and accuracy includes chemiluminescent immunoassay (CLIA) and time-resolved immunofluorometric assay (TR-IFMA). These methods generally enhance sensitivity and provide a broader dynamic range than the traditional ELISA. Soon after the development of the first IGF-I RIA it was realized that measurement of IGF-I (and IGFII) requires dissociation of IGFs from IGFBPs prior to assay. Size exclusion chromatography (often performed as fast protein liquid chromatography, FPLC) at low pH is considered the gold standard [2], but due its laborious nature, less optimal methods that were more suitable for large scale assays were developed. Generally, these methods were based on acidification of serum, followed by precipitation of the IGFBPs using an organic solvent [60]. However, these methods failed to remove all IGFBPs while IGFs were co-precipitated during the extraction procedure. They therefore contained an inherent risk of yielding falsely reduced levels. Therefore, in most commercial assays this problem is usually managed by the use of a low pH reagent that dissociates IGF-I from the IGFBPs, whereafter excess IGF-II is added to prevent reassociation of the IGFBPs with IGF-I [2,51].

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IGF assay pitfalls Measurement of IGF-I represents a cornerstone in the diagnosis and treatment of pituitary diseases and growth disorders as well as in research areas covering metabolic disorders and type 2 diabetes, cancer, longevity as well as neurological and cardiovascular diseases [17,61–64]. However, the comparability of results, and in particular of clinically relevant cut-off limits, has been hampered by differences in IGF-I concentrations obtained by various assays. These differences are primarily caused by variable assay characteristics. As a consequence, a consensus statement has been made with the purpose of improving harmonisation of IGF-I assays [2]. Differences in reported IGF-I values between assays may relate to the use of different reference preparations of IGF-I. Therefore, the consensus statement advocates the use of a common IGF-I standard; the first international standard for IGF-I, encoded WHO 02/254. This standard consists of highly pure recombinant human IGF-I, which has been thoroughly validated and tested in more than 18 different laboratories in nine countries [65]. Implementation of this IGF-I standard constitutes a significant step towards international harmonization of IGF-I assays. The standard is available from the National Institute for Biological Standards and Control (NIBSC; homepage: http://www.nibsc.ac.uk) and as the prize is reasonable, we see no reason not to calibrate all IGF-I assays against WHO 02/254. Having said this, it is important to stress that use of WHO 02/254 may not completely eliminate between-assay differences. Thus, Krebs et al. demonstrated large differences between five commercial IGF-I assays despite the fact that they were all calibrated against the same (old) IGF-I reference standard WHO 87/518 and that some of the assays used the same principle (IGF-II excess) to block IGFBP interference [66]. An important outcome of that study was that for some of the tested assays, the correlation was sufficiently strong to allow conversion of IGF-I values from one assay to the other, whereas for other assays, the relationship was too poor to justify this approach. Since the assays were calibrated against the same standard, the between-assay differences can only be explained by differences in antibody specificity and/or IGFBP interference. On this basis, the consensus statement does not encourage the use of conversion factors between assays, although this approach may be necessary during a transition period [2]. Blocking of interfering IGFBPs with IGF-II circumvents the problems experienced with older extraction methods [60], where the IGFBPs were dissociated at low pH and subsequently precipitated using an organic solvent and separated by centrifugation. In these methods, incomplete IGFBP removal and co-precipitation of the IGFs were an inherent risk, in particular in “tricky” serum samples from patients with highly abnormal IGFBP levels. However, the IGF-II blocking principle is not flawless. The efficacy of excess IGF-II to block IGFBP interference is related to the ratio between IGF-I and IGF-II, as well as the concentration of unsaturated IGFBPs. The latter may be a particular problem in diseases such as anorexia nervosa [8,67], type 1 diabetes [68], chronic renal failure [69,70] and liver cirrhosis [71]. These conditions are characterized by increased IGFBP levels, in particular IGFBP-1 and -2, and as the endogenous IGF-concentrations range from very low to normal, the concentration of unsaturated IGFBPs is likely to be higher than normal. Under such circumstances a higher excess of IGF-II may be required to block IGFBP interference. Kit manufacturers should document that their IGF-II blocking method is indeed able to facilitate accurate measurements in problematic samples, and the end user should confirm that this information is indeed stated in the kit insert. Endogenous heterophilic antibodies are known to interfere in immunoassays, and although this is likely to represent a relatively infrequent problem in the context of IGF-I and its related proteins, its precise magnitude is hard to estimate. So far, a single case report has described the presence of heterophilic antibodies, which resulted in falsely low IGF-I levels causing suspicion of GH deficiency [72]. Recently, auto-antibodies against IGFBP-2 have been detected in patients suffering from pulmonary disease, in particular malignancies, and although these auto-antibodies appear to be relatively frequent, their ability to interfere with IGFBP-2 immunoassays is less certain [3]. There is no consensus regarding determination of IGF-II, but obviously, its measurement is encumbered with interference from the IGFBPs. In contrast to IGF-I, circulating IGF-II is composed of different molecular forms. IGF-II is synthesized as pre-pro-IGF-II, which is composed of an N-terminal 24 amino acid signal sequence, the 67 amino acid mature IGF-II and a 90 amino acid E-domain at the C-

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terminal. Pro-IGF-II is formed after cleavage of the signal protein, whereafter the E-domain is removed prior to secretion. However, due to alternative cleavage sites on the E-domain, some of the secreted IGF-II may still contain parts of the E-domain. These forms are referred to as big IGF-II [73]. We measure pro- and big-IGF-II using an antibody directed against amino acids 78–88 [74]and find these forms to constitute about 27% of the total IGF-II reactivity when our mature IGF-II assay is calibrated against the WHO 99/538 reference standard (unpublished observations). These values are within the range reported by others [73]. As the larger forms of IGF-II only contain an extension of the mature peptide, it is to be expected that many assays for mature IGF-II also recognizes pro- and big-IGF-II. However, this aspect is seldom taken into consideration. Immunoassays for determination of IGFBPs The IGFBPs were first identified by virtue of their ability to bind IGFs. Their presence was revealed by gel filtration chromatography, in which the IGFs migrated in high-molecular complexes of approximately 40 and 150 kDa. Later, Hossenlopp et al. used the Western ligand blotting (WLB) technique to detect and quantify the IGFBPs [75]. In the WLB assay, the IGFBPs are separated according to size by SDS-PAGE, electrotransferred to a nitrocellulose sheet and detected using iodinated IGF-I or -II. Since proteolytic fragments of IGFBP have a very low affinity for the IGFs, WLB only detects intact IGFBPs. As an alternative, the Western immunoblotting technique (WIB) [76] uses specific antibodies to probe for the IGFBPs and detects both intact and fragmented forms. Although these methods are neither very quantitative nor specific, they have been widely used for detection and visualisation of the IGFBPs in all kinds of fluids. The development of specific RIAs and IRMAs further improved IGFBP research and the increased sensitivity also allowed for reliable determination of low concentrations of IGFBPs. At the time of writing, numerous assays, commercially as well as non-commercially have been developed, and due to their easy accessibility, it is uncommon for an IGF-I study not to report data on at least some of the IGFBPs, most frequently IGFBP-1 to -3. Pitfalls and challenges of IGFBP assays As previously mentioned there is no consensus for the measurement of IGFBPs. However, IGFBP immunoassays are complicated by numerous pitfalls, the most important being the presence of immunoreactive IGFBP fragments, which may be co-measured indiscriminately from full length IGFBPs, resulting in erroneous interpretations. In contrast to IGF-I and IGF-II, all IGFBPs are prone to proteolytic digestion, markedly compromising their ligand affinity. Enzymatic cleavage of the IGFBPs is actually believed to play a key role in liberating bound IGF [10,12,42]. Proteolysis of IGFBP-3 may serve as an example illustrating how this can affect immunoreactivity. IGFBP-3 is extensively proteolyzed during pregnancy, being almost completely degraded at 8 weeks [77], as well as during sepsis, severe systemic disease and following major surgery [78]. However, whereas proteolyzed IGFBP-3 in pregnancy retains its ability to bind IGF-I in ternary complexes, this is not the case in the other conditions [79]. Thus, pregnancy is associated with normal to high IGF-I levels while sepsis and surgery is associated with low IGF-I levels [77,79]. The discrepant impact of proteolysis on IGFBP-3 is most likely explained by the involvement of different proteases and consequently different cleavage sites. As the vast majority of IGFBP-3 assays recognize IGFBP-3 fragments, albeit most likely with different affinity, this may affect the measured IGFBP-3 concentration in an assay-specific manner. Two different immunoassay approaches have been developed to take into account the presence of IGFBP fragments. The first approach is assays that only detect IGFBPs that are able to bind the IGFs, i.e. functional assays. This principle was employed by Langkamp et al., who developed an immunofunctional assay which only detects IGFBP-3 being able to interact with biotinylated IGF-I [80]. The other principle is based on the development of antibodies specific for intact IGFBP-3. This approach was employed by Koistinen et al. [81,82], who developed two TR-IFMAs based on monoclonal antibodies; one specific for intact IGFBP-3 and one which co-measured many of the IGFBP-3 fragments. Such assays are likely to yield novel information on the fragmentation of IGFBPs in normal and pathophysiological conditions, whereas it remains to be clarified whether they have any clinical advantages over traditional IGFBP-3 immunoassay.

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It should be remembered that the IGFBP-degrading enzymes may also be operational after the blood sample has been collected, in particular when serum is prepared. Additionally, enzymatic degradation of the IGFBPs may be an issue when samples are undergoing repetitive freezing and thawing. If the assay is specific for intact (full length) protein only, or only recognizes the fragments with a low affinity, this may affect the measurable concentration of IGFBP significantly. Thus, this aspect should be taken into consideration when planning the assay sequence. IGF-I and IGF-II are usually very stable even when exposed to repetitive freezing and thawing, most likely because they are not degraded as easily as the IGFBPs [51,52,83]. The kidneys partake in the clearance of circulating peptides and therefore, impaired kidney function as seen in chronic renal failure (CRF) results in accumulation of degraded, low molecular weight IGFBP fragments. Many of these fragments retain some affinity for IGF-I and this may explain why patients with CRF may have normal to high IGF-I levels in spite of low levels of free and bioactive IGF-I [8,70,84]. Interestingly, when measuring IGFBP-3 in CRF serum, levels are normal to high when measured by immunoassay, whereas WIB show normal levels of intact IGFBP-3. Thus, high levels of IGFBP-3 in serum from CRF patients appear to be caused by accumulation of immunoreactive IGFBP-3 fragments. It is well recognized that the IGFBPs are prone to undergo post-translational modification, predominantly phosphorylation and glycosylation [12]. The physiological significance of these posttranslational modifications remains, however, speculative, and it is not either known to which extent such modifications affect antibody recognition. An exception is IGFBP-1, which exists in nonphosphorylated as well as in highly phosphorylated isoforms. It is well-known that different assays for IGFBP-1 may yield highly different values (up to a factor of 10 between assays) and this appears to relate to the ability of the antibodies to detect phosphorylated isoforms [77,85]. The IGFBPs exist either as free IGFBP or complexed to IGF-I or IGF-II in a 1:1 M ratio. However, traditional immunoassays do not discriminate between IGFBP and IGFBP:IGF complexes. Although this apparently is no problem for the clinical interpretation of an IGFBP result, it clearly limits the understanding of how the IGFBPs take part in the control of IGF-action. Establishment of assays specific for IGFBP:IGF-complexes have been described by us [86] and others [87]. Our assay was specific for complexes composed of IGF-I:IGFBP-1 and by that we were able to show that an increase in IGFBP-1 during fasting was parallelled by an increase in the IGF-I:IGFBP-1 complex, resulting in reduced levels of free IGF-I [86]. Thus, we believe that much knowledge on the dynamics between the IGFs and their IGFBPs can be obtained by refining the IGFBP assays. Alternative methods for determination of IGF-I The worldwide interest in IGF-I have brought along a number of alternatives to the conventional immunoassays. Modified assays for the measurements of free [8] and bioavailable IGF-I [88] have been developed with the purpose to improve estimations of the endogenous IGF bioactivity. Of certain interest has been the development of the kinase receptor activation (KIRA) assay, a methodology which was originally described by Sadick et al. [89] and later refined in our laboratory [88]. The KIRA assay uses cells transfected with the IGF-IR and measures the ability of IGF-I in a sample to activate the receptor protein kinase in vitro, leading to receptor phosphorylation. Subsequently, the concentration of phosphorylated receptors is determined in a conventional immunoassay. Although the method setup may not represent in vivo like conditions, it reflects the IGF activity in a given sample, and data so far have yielded novel information. For example, we have shown that the in vitro IGF-I bioactivity in ascites and in extracellular fluids collected with the suction blister technique may be higher than in the corresponding serum samples [11,90]. However, due to the laborious nature of such bioassays, we doubt that they will gain broad clinical relevance, whereas we are confident that they may be valuable by improving our understanding of the IGF system. Proteomics, and especially mass spectrometry (MS), have for several years been used for identification and characterization of proteins, and they now offer an attractive alternative to present immunoassays for quantitative analysis of the IGF system. Mass spectrometry possesses an enormous discriminative power and allows for determination of specific proteins with high sensitivity and without any cross-reactivity or interference. Within the last decade, MS has crossed over from the field of descriptive chemistry into the area of clinical medicine and quantitative analysis of IGFs using this

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method has increased and improved [91,92]. Recently, MS has been applied in various clinical studies [93,94]. Compared to conventional immunoassays, the MS approach allows for determination and discrimination of different IGF species, e.g. intact IGF and its truncated metabolites and provides low limits of detection. MS has also been used to detect exogenously administered recombinant human IGF-I used for doping purposes [95]. However, MS still requires disruption of the IGF/IGFBP complex prior to measurements, and the potential loss of IGF during the extraction step still represents a problem in MS. Summary Accurate measurements of IGFs and IGFBPs are necessary to obtain information about their physiological roles and potential implications in human pathologies. Immunoassays are the main approach to determine circulating levels of IGFs and IGFBPs, but as immunoassays rely on the specific binding of the antibody to a distinct tertiary structure on the target protein, several technical as well as biological factors are able to modify and alter immunoreactivity. These factors are not always easily identified, and may require detailed and skilled assay validation. Therefore, there is a need for standardized validation procedures in order to provide reliable and comparable results, allowing for direct comparisons across studies. Also, further development of novel assay strategies and improvement of reference materials are required, as well as standardization of sample collection and handling. This would lead to better assay performance and comparability of data.

Practice points  The measurement of IGF-I should be performed according to the 2011 consensus statement, i.e., the assay should be calibrated against the WHO IGF-I reference preparation 02/254, and interference from the IGFBPs should be taken care of by blocking with excess IGF-II.  For commercial IGF-I kits, the assay should be thoroughly validated by the manufacturer and this information should be made available to the users.  In order to determine whether a patient sample contains a normal or abnormal IGF-I level, the clinicians need access to comprehensive assay specific reference ranges.  In general, reference values are assay specific and the use of correction factors between different assays should be used with great caution.

Research agenda  There is a need to consider creating consensus statements for measurement of the various IGFBPs as well as IGF-II.  In regards to IGF-II there is a particular requirement for developing assays with and without the ability to co-measure pro- and big-IGF-II as this may promote insight into the role of IGF-II in human physiology and pathophysiology  Continued development of alternative IGF and IGFBP assays, for instance assays measuring biological activity in vitro as well as assays specific for unsaturated and saturated IGFBPs. Such assays may be helpful to increase our knowledge on the roles of the IGFBPs in health and disease.

Acknowledgement Dr. Rikke Hjortebjerg and Professor Jan Frystyk have been funded by Department of Clinical Medicine at Faculty of Health, Aarhus University, Denmark.

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