IGF-1 and IGFBP 3 in Growth Hormone Deficiency Role of Insulin Like Growth Factor-1 ( IGF-1 ) and IGF Binding Protein 3 in the Diagnosis of Growth Hormone Deficiency : Changing Paradigm

GH stimulation tests are widely used in the diagnosis of GH deficiency (GHD), although they are associated with a high false positive rate. Serum IGF-I levels are monitored during GH replacement treatment in subjects with GH deficiency (GHD) to guide GH dose adjustment and to minimize occurrence of GHrelated side-effects. The need for reliance on provocative testing is based on evidence that the evaluation of spontaneous growth hormone (GH) secretion over 24 hours and the measurement of IGF-I and IGFBP-3 levels do not distinguish between normal and GHD subjects. Regarding IGF-I, it has been demonstrated that very low levels in patients highly suspected for GHD (i.e., patients with childhood-onset, severe GHD, or with multiple hypopituitarism acquired in adulthood) may be considered definitive evidence for severe GHD obviating the need for provocative tests. However, normal IGF-I levels do not rule out severe GHD and therefore adults suspected for GHD and with normal IGF-I levels must undergo a provocative test of GH secretion. We hereby review the various literatures at disposal justifying the use of IGF-1 and IGBP3 for diagnosis of growth hormone deficiency. Data Source: We searched PUBMED and MEDLINE database for relevant articles including key words. References of each article were further reviewed for final synthesis of the manuscript.


Introduction
D iagnosis of growth hormone (GH) defi ciency (GHD) is still a matter of debate.Assuming that no clinical feature can be relied on as a guide, the approach to diagnosis must include a high index of suspicion of hypothalamic-pituitary disease.At present, the insulin tolerance test (ITT) is the diagnostic tool of choice 1 , even though this test is contraindicated in patients with electrocardiographic evidence or history of ischemic heart disease or in patients with seizure disorders.Other stimulation tests have proved their usefulness for GHD diagnosis but the maximum response for GH diagnosis has not been unanimously established in an adult population 2 .
Insulin-like growth factors (IGFs) are polypeptides that act as endocrine mediators of growth hormone (GH)-induced anabolic and mitogenic actions.They also function in a paracrine and autocrine manner to regulate cell growth, diff erentiation, apoptosis and transformation.The IGF system is a complex network comprised of two growth factors (IGF-I and -II), cell surface receptors (IGF-IR and -IIR), high affi nity binding proteins (IGFBP), IGFBP proteases as well as several other IGFBP-interacting molecules, which regulate and propagate IGF actions in several tissues 3 .The clinical use of measurements of IGF-I has been focused primarily on diagnosing or excluding GH defi ciency (GHD) and monitoring GH therapy.
IGF-I and IGF-BP3 are being widely used for evaluation of the diagnosis of GHD.Owing to the limitations of GH stimulation tests, there has been a gradual shift from GH-based approaches to those utilizing IGF for the diagnosis of GHD 4 .However, the use of IGF-l or IGF-BP3 in the diagnosis of GHD in children is a matter of controversy 5,6 because of variable sensitivity and specifi city of these tests 5,7,8 .
Factors infl uencing levels of IGFs are age, sex, pubertal status, nutritional status, diabetes mellitus, renal failure and liver functions 9 .A reduced IGF-I level may occur in a child with malnutrition 10 , hypothyroidism 11 , hepatic disease, or diabetes mellitus 12 , as well as GHD.Pulsatile growth hormone (GH) secretion stimulates GH-responsive issues to produce IGF-I.IGF-I in plasma is primarily derived from the liver and circulates bound to specifi c IGF-binding proteins (IGFBPs), six of which (IGFBP 1-6) have been characterized.Most (99%) IGF-I circulates bound to IGFBP-3 in l50-kDa complex.Serum IGFBP-3 concentrations are directly proportional to GH concentrations and nutritional status.IGFBP3 is considered a good marker for the GH-IGF axis.
Both IGF-1 and IGF BP 3 are GH-regulated, but ageand nutrition-dependency often renders interpretation of results diffi cult, thus making it desirable for each laboratory to have its own normative data matched for age and body mass index (BMI).However, such data are not always available since their collection is expensive, time consuming and requires a high degree of collaboration between clinical and laboratory departments.Thus, it is common practice to make use of reference values provided by commercial assay manufacturers, which include values obtained in a group of control subjects, usually stratifi ed by age and/ or sex, but lacking any information on body fatness measurement.Altered body composition, with increased body weight and body fat mass and decreased lean body mass, is a clinical characteristic of GHD and should be taken into account in the matched reference population Rosenfeld, et al have found that18% of the putative GH defi cient children had serum IGF-1 levels within the normal range for age, and 32% of normal short children had low IGF-1 levels 4 .

The role of IGF-l in the diagnosis of growth hormone deficiency (GHD)
GH secretion can either be measured through investigation of the pituitary or by monitoring markers that change as a consequence of GH action on its target tissues.The two most widely used and best-validated biochemical parameters are immunoassay measurement of either GH or IGF-I.The fi rst refl ects GH secretion while the second refl ects GH action.Since GH secretion is pulsatile in nature 13 , GH provocative/stimulation tests are essential.

GH stimulation tests have many fallacies:
1.The insulin tolerance test has been considered the gold standard for the assessment of GH axis 14 .However, it has been associated with mortality and morbidity in children due to associated hypoglycemia.
2. There is no consensus as to which of the other agents are most suitable.The sensitivity and specifi city of arginine and clonidine stimulation tests are 73% and 85%, and 70% and 85% respectively 6 .There is no agreement on the cut-off GH levels for each assay (i.e., 7 or 10 ng/ mL) to defi ne normality.
3. There is a problem of reproducibility and the tests are associated with a wide coeffi cient of variation 15 .
4. The most signifi cant drawback of these tests has been the lack of normative data.Stimulated GH levels have little resemblance to the growth dynamics of some normal children 6 .
5. Prepubertal children with normal stature may fail to attain peak GH values more than 7 μg/L during GH provocative test 6 .In a study by Marin, et al. 16 , there was a high incidence of peak GH concentration consistent with GH defi ciency among normal children, i.e. 61 % in the prepubertal children had a GH peak less than 7 μg/L.
With advancing puberty, the percentage of children with normal stature who failed to attain a GH level greater than 7 μg/L in response to arginine, insulin and standardized treadmill exercise declined from 61% at pubertal stage 1 to 44% at stage 2, 11% at stage 3, and 0% at stages 4 and 5. Administration of estrogen to the prepubertal subjects raised the normal range for the peak GH response to the three tests.Thus, both puberty and estrogen administration signifi cantly increase the peak GH response to exercise, arginine, or insulin in normal subjects.
Owing to the limitations of GH stimulation tests, there has been a gradual shift from GH-based approaches to that utilizing IGF for the diagnosis of GHD 4 .The serum level of the major GH-dependent peptide IGF-I is stable during the day, due mainly to the complexing of IGF peptides with a family of IGF-binding proteins (IGFBPs) 17 .Thus the potential for assessing GH status with a single estimation of the circulating IGF-I level proved attractive Kota S K et al and gave rise to the hope that eventually dynamic GH provocation tests may become unnecessary.Initially IGF assay problems were caused by interference from the presence of IGFBPs 18,19 .This problem has been overcome by using a variety of approaches including acid size exclusion chromatography before the IGF assay, blocking IGFBP-binding sites with an excess of the unmeasured IGF peptide (excess IGF-II for an IGF-I assay) or IGF analogs that do not bind to IGFBPs as radioligands 10 .
The sensitivity and specifi city of IGF-I in the diagnosis of GH defi ciency in children is a matter of controversy because: 1.The liver is the principal source of IGF-I in the circulation 2. Hepatic production of IGF-I is highly infl uenced by nutritional factors 3. It is possible that decrements in IGF-I expected with GHD are modifi ed by nutritional status and other factors, such that only severe GHD produces a clear segregation of children who are defi cient from those who are not.
4. IGF-I levels are not only aff ected by age and nutritional status but also by thyroid hormones and gonadal steroids.Therefore, the patient should be euthyroid and pubertal staging should be assessed prior to estimation of IGF-I.The IGF-I level is infl uenced markedly by age and pubertal development 20,21,22 .Particularly low concentrations of IGF-I occur in normal children younger than 5 yr of age.Therefore, the use of IGF-I estimation to distinguish between normal and GH-defi cient children is less successful in this age group.
Nevertheless the measurement of serum IGF-I is useful since it can be derived from a single blood sample and checked frequently during evaluation, monitoring and treatment of a child with growth failure.Since IGF-I levels vary with ethnicity, it is important to generate population specifi c normal ranges through childhood and adolescence, incorporating all pubertal stages 7,8 .There is a paucity of Indian studies on IGF-I and IGF-BP3.Dehiya, et al. 17  Recently, it has been argued that total IGF-I concentration, in analogy with thyroid and sex steroid hormones, may not refl ect IGF bioactivity.Thus, measurement of the serum unbound IGF-I fraction (free IGF-I) might have greater physiological and clinical importance than its total concentration 23 .The ratio on a molar basis between IGF-I and the predominant IGF binding protein (IGFBP-3) has been reported to correlate with free IGF-I 24 and has been proposed as an indirect measure of the easily dissociable circulating IGF-I 25 .
The performance of IGF-I has mostly been evaluated in children diagnosed as GH defi ciency on basis of short stature, poor growth velocity and sub-optimal GH levels during 2 provocative tests.Blum, et al. 7 found sensitivity and specifi city of IGF-I to be 92% and 54% respectively, using their normal ranges, when applied to their subjects (mean age 11.2 years) with GH defi ciency.The peak GH level was 10 ng/mL (in response to both arginine and insulin).Such a high degree of sensitivity has not been found in other studies.Poor specifi city was related to the fact that low IGF-I levels are relatively common in normal prepubertal children.There are still doubts about which is the most appropriate cut-off line for patients with GHD.Moreover, misclassifi cation of subjects may occur, while taking into account false positive and negative results during GH stimulation tests.Diff erent researchers have used cut-off lines based on standard criteria, such as the 5 th percentile, the 10th percentile, or 2 SD in relation to the mean.Based on these criteria, a performance of IGF-I has been reported in children in the diagnosis of GHD with a sensitivity ranging from 34-100% and a specifi city of 47-99% in diff erent studies 5,7,8,[26][27][28][29] .Table 1 summarizes the specifi city and sensitivity of IGF-I in various studies.Using GH testing as the gold standard to distinguish between 155 children with GHD and 219 with normal short stature, an IGF-I estimation at the fi fth percentile level of the normal range provided a sensitivity of 95%, specifi city of 60%, and accuracy of 75%, while improved fi gures of 79%, 95%, and 89%, respectively, were associated with an IGF-I cut-off level of 0.1 centile 30 .The IGF-I results discriminated better in those over 8 IGF-1 and IGFBP 3 in Growth Hormone Deficiency ... yr compared with those under 8 yr of age 30 .Juul and Skakkebaek 31 also concluded that measurement of IGF-I was useful in the diagnosis of childhood GHD but, unlike the fi ndings reported by Blum 30 , more so in younger than older children.They reported accuracy (predictive value) of 88.8% and 52.3% in children aged less than 10 yr and between 10 and 20 yr, respectively 31 .In the latter studies 30,31 the IGF-I values were corrected for age and sex but not pubertal status.
Despite the promise of these results, multiple studies exist that indicate the serum IGF-I concentration does not correlate perfectly with GH status, as determined by provocative GH testing 32,33,34 .To improve the diagnostic value of IGF-I measurements, the additional measurement of IGF-II has been recommended 35 .Despite the fact that IGF-II is less GH dependent than IGF-I, the normal range for IGF-II levels is relatively high in young children and shows little age dependence.Thus, although the accuracy of serum IGF-II measurement alone in detecting GHD is moderate, the combined use of IGF-I and IGF-II improves their value as a diagnostic parameter.In a study of 68 GH-defi cient children, 197 normal-statured children, and 44 normal short children, 18% of the GH-defi cient children had serum IGF-I levels within the normal range, whereas 32% of normal short children exhibited low IGF-I concentrations.Low IGF-II levels were found in 52% of GH-defi cient children but also in 35% of normal short children.Utilizing the results from both IGF assays, however, revealed that 4% of GHdefi cient children had normal IGF-I and IGF-II levels but only 0.5% of normal children and 11% of normal short children had reduced concentrations of both IGF-I and IGF-II 35 .In reality, however, very few paediatric endocrine centres use IGF-II assays in the assessment of children with short stature.Thus, low levels of IGF-I may be indicative of GHD.The plasma concentrations of the IGF-I could be considered useful indicators of GH bioactivity in children when correlated clinically, and accounting for other confounding factors.However, it is noteworthy that a normal serum IGF-l level does not exclude the presence of GHD.

Role of IGF-BP3 in the diagnosis of GH deficiency (GHD)
Of the six known IGFBPs, IGFBP-3 is normally the major serum carrier of IGF peptides 36 .IGFBP-3 circulates as part of a ternary complex consisting of IGFBP-3, an IGF peptide, and an acid-labile subunit 37,38 .Both acidlabile subunit and IGFBP-3 are GH dependent.Since IGFBP-3 determinations refl ect combined IGF-I and IGF-II concentrations, age dependency of IGFBP-3 is less striking than for IGF-I.
Since IGFBP-3 serum levels are constant throughout the day and are closely GH dependent, it was proposed as a reliable and simple screening test in the work-up of children with short stature and preliminary results were promising.Blum, et al 7 observed the sensitivity and specifi city of IGF-BP3 to be 97% and 95% respectively.Moreover, IGF-BP3 measurement off ers several important advantages over IGF-I determination 28 : 1.No extraction step is required before measurement, thereby improving the precision and facilitating the procedure 2. IGFBP-3 normally circulates in the serum at high concentrations, so that assay sensitivity is not an issue.
3. IGFBP-3 serum concentrations, like IGF-I, are agedependent, but the normal range varies only modestly with age and pubertal status.
4. The impact of nutritional status is not as signifi cant as with IGF-I.
Several studies have addressed the issue of sensitivity and specifi city of IGFBP-3 assessment in the diagnosis of GHD, yielding confl icting results 5,7,8,[26][27][28][29]39 (Table 2). In more recent evaluation of the usefulness of IGFBP-3 measurement in the diagnosis of GHD, Juul and Skakkebaek 31 reported sensitivity and specifi city fi gures of 60% and 97.9% in children less than 10 yr of age and 56.5% and 78.7%, respectively, in those aged between 10 and 20 yr.These results could not be reproduced in other studies 32,[40][41][42][43] .Furthermore, the IGFBP-3 estimation discriminated particularly poorly between GHD and Kota S K et al normality in pubertal children and those with radiationinduced GHD 32,44 .
Cianfarani et al reported poor sensitivity of IGFBP-3 evaluation, suggesting that proteolysis is likely to aff ect IGFBP-3 assay results 39 .Poor sensitivity of IGF binding protein (IGFBP)-3 assessment in the work-up of GH defi ciency (GHD) has been ascribed to IGFBP-3 proteolysis 16 .On the other hand, specifi city of IGF-BP3 for the diagnosis of GHD has been generally reported by various studies to be high 5,7,8,[26][27][28] .
It has been proposed that the measurement of the IGFBP-2 concentration adds to the value of IGF-I and IGFBP-3 assays in the diagnosis of GHD 45 .IGFBP-2 values are usually elevated in patients with GHD 45,46 .Despite the fact that in most patients there is agreement between the IGFBP-2/ IGF-I ratio, IGFBP-3 measurements, and the results of GH testing, the IGFBP-2/IGF-I ratio was discordant from thigh response in 21% of 80 patients (57 GHD and 23 idiopathic short stature), and IGFBP-3 results were discordant in 18% 45 .
The proponents of IGF-I and IGFBP-3 estimations as the best choice of initial investigations in a child with possible GHD point out that in individuals with genetic forms of GH insensitivity (GHI), all have a low and most have a low IGF-I concentration 47,48 .By defi nition, these individuals have elevated GH concentrations associated with a mutation or deletion of the gene for the GH receptor, which renders them insensitive to GH action.Furthermore, despite the universally low IGFBP-3 concentrations and the characteristically severe growth failure, serum IGFBP-3 concentrations still correlate signifi cantly with the height SDS 47,49 .
Thus, low levels of IGF-BP3 are very specifi c for the diagnosis of GHD, indicating its clinical utility.However, due to poor sensitivity of IGFBP3, normal serum level does not exclude GHD.

IGF-1 and IGF-BP3 in the diagnosis of Adult GHD
The changes in IGF-I levels throughout life are similar to those of GH.With the onset of puberty there is a 2 to 3-fold rise in serum IGF-I concentrations followed by a decline such that average adult levels are reached by the early twenties 22 .There follows a gradual decline with advancing age 22 .Similar to IGF-I but less age-dependent, serum IGFBP-3 levels rise to a peak during the pubertal years and then slowly decline in adulthood 49 .Thus, in the adult patient with potential GHD, serum IGF-I and IGFBP-3 measurements can only be interpreted if decadebased normative data are available.The usefulness of an IGF-I estimation in the diagnosis of adult GHD is a matter of contention although it has become clear that at least some of the disparity between studies is explained by the timing of the onset of GHD.Hoff man et al. 50found that 70% of IGF-I and 72% of IGFBP-3 values in adultonset GHD patients, mean age 45 yr, were within the range of normal subjects even allowing for the eff ects of age.Holmes 51 , in a subsequent study of 65 adults, mean age 35 yr, with GHD defi ned by a peak GH response of less than 3.8 ng/ml to a provocative test, and associated with other evidence of pituitary disease, found that 70% had an IGF-I SDS below 22.In the latter study, however, the 65 adults consisted of a mixture of childhood-onset and adult-onset GHD 51 .DeBoer et al. 52 focused on childhood-onset GHD and reevaluated GH status in 89 young adult males who had previously received GH replacement in childhood.Approximately 93% of the patients had an IGF-I level below the normal range with a similar number of subnormal IGFBP-3 levels among the patients (Fig. 1).Attanasio et al. 52 pursued the same theme in a large study of 74 childhood-onset and 99 adult-onset GHD patients.They concluded that there are profound diff erences between these two forms of GHD 53 .With group data they observed that the serum IGF-I levels were below normal in both groups of GH-defi cient patients but were signifi cantly lower in childhood-onset than adultonset GHD patients 53 .Other authors have made similar observations 54,55 .In these studies, however, it is not clear whether suffi cient decade-based normative data were available or whether the severity of GHD was equal in the childhood-onset and adult-onset groups.Thus, apart from the potential impact of the timing of onset of GHD, it is possible that the severity of GHD infl uences the interpretation of IGF-I results.
To pursue this question further, Shalet et al 56 have analyzed the IGF-I SDS results of the mixed population of 65 adults with childhood-and adult-onset GHD studied by Holmes 51 in the context of the severity of GHD.A direct comparison of the IGF-I SDS with peak GH response was impossible due to the multiplicity of GH provocative tests used in that study 51 .Therefore, the IGF-I SDS data was stratifi ed, in the context of additional pituitary hormone defi cits, into those with GHDO, GHD1, GHD2, and GHD3 (Fig. 2).In each of the latter categories, there was a mix of adult-onset and childhood-onset GHD patients, thereby allowing a comparison of IGF-I status among groups with tightly defi ned degrees of GHD.The numbers were suffi cient for statistical comparison in the GHDO, GHD1, and GHD3 groups and, in each category, the median childhood-onset IGF-I SDS is lower than the median adult-onset IGF-I SDS, reaching signifi cance in two of the three groups (GHDO,23.83vs.22.69,P50.239; GHDI, 25.75 vs. 22.03,P 5 0.022; GHD3, 26.28 vs. 22.09,P50.0003) 51 .These observations suggest that for a given IGF-1 and IGFBP 3 in Growth Hormone Deficiency ... degree of GHD, the serum IGF-I concentration is lower in adults with childhood-onset compared with adultonset disease.The explanation for this latter observation is unclear; however, it supports the notion that an IGF-I estimation is more likely to be of diagnostic use in childhood-onset than in adult-onset GHD.
An additional observation needs to be made in the patients with adult-onset isolated GHD.Toogood et al. 57 had shown previously that 24% of GHDO patients had a peak GH response of less than 1.9 ng/ml to an ITT.In the data of Holmes 51 , there are 11 patients in the GHDO group with adult onset disease, eight of whom have an IGF-I SDS below 22.Therefore, any therapeutic strategy to decide which GH defi cient patients warrant a trial of GH therapy must allow for patients with isolated GHD, who on an individual basis may show as severe a degree of GHD as a patient with panhypopituitarism.

Rational approach to the diagnosis of GHD
Normal levels of IGF-I and IGF-BP3 do not exclude a diagnosis of GHD.The high specifi city of IGF-I and IGFBP-3 measurements suggests that while a combination of a low IGF-l and low IGFBP-3 would be highly suggestive of GHD, signifi cant number of children with GHD will have normal values for either of these two markers.Mitchell, et al. 28 have observed that, if, for a diagnosis of GHD, the requirement were for both these tests to be positive, then 99% of children without GHD would be correctly identifi ed; however, the sensitivity of this test was only 15%.Hence, neither IGF-I nor IGFBP-3 alone is a surrogate marker for GHD nor even when analyzed in combination, they cannot be used as surrogate markers for GHD.Tillmann, et al. 5 devised a scoring system for diagnosis of GH defi ciency based on the positive predictive value of the GH stimulation test, and the IGF-I and IGFBP-3 levels.A high score was highly indicative of GHD, but was achieved by few patients.A normal IGFBP-3 level, however, did not exclude GHD.GH stimulation test with a peak level more than 10 ng/mL was the most useful single investigation to exclude a diagnosis of GHD.
Peak GH response to two diff erent provocative tests less than 7 or 10 ng/mL has been considered previously as essential for the confi rmation of GHD 6 .However, in a study by Cianfarani, et al. 58 , a simple assessment of height velocity (HV) and basal IGF-I in association with only one GH stimulation test, has been shown to confi rm the diagnosis of GHD in a majority of patients.
It is useful to schedule IGF-I and IGF-BP-3 to study abnormalities of GH-IGF axis, and as part of initial screening and diagnostic workup in short children.This should be followed by GH provocative tests for making a defi nitive diagnosis of GHD 59 .Thus a rational diagnostic approach to the diagnosis of GH defi ciency should emphasize good history and auxologic measurements, evaluation of IGF-I and IGF-BP3 levels to identify GH-IGF axis abnormalities, and fi nally confi rmed by GH provocative tests 60 .

Conclusion
Thus, low IGF-I and IGFBP-3 concentrations are reliable guides to the diagnosis of severe GHD, provided the investigator considers the alternative possibilities of malnutrition, hypothyroidism, liver disease, and GHI.Discrepancies between IGF-I and IGFBP-3 results compared with the GH responses to dynamic tests exist, however, in those children believed to have lesser degrees of GHD.The inadequacies of dynamic provocative tests of GH release, outlined previously, may explain the discrepancies in the GH status defi ned by the GH and IGF axis results.Alternatively, it may be that IGF-I and IGFBP-3 estimations are less useful than the peak GH response to provocative testing in children with GH insuffi ciency.
have analyzed levels of IGF-I and IGF-BP3 in healthy children and adolescents (from birth to 20 years of age) residing in Mumbai suburbs.

Table 1 :
The performance of IGF-1 in the diagnosis of Growth hormone defi ciency

Table 2 :
Performance of IGF BP 3in the diagnosis of Growth hormone defi ciency