Probing the GH–IGF Axis
Laboratory Support for Growth Disorders
by Martin W. Elmlinger, Ph.D.
Assistant Professor in Clinical Chemistry, Endocrinology
University Hospital, Tübingen
Tübingen, Germany

and Werner Kühnel, Ph.D.
Marketing Manager, Scientific Affairs
DPC Biermann, Bad Nauheim, Germany

In the diagnosis and management of conditions associated with growth hormone (GH) deficiency or excess, the pulsatile secretion of this pituitary hormone severely limits the information one can expect from a single determination of GH based on a routine ("random") blood draw. Accordingly, for direct laboratory assessment of somatotropic function, endocrinologists rely primarily on provocative testing of GH secretion and on the determination of spontaneous GH-secretion profiles integrated overnight, or over 24 hours.

Assays for insulin-like growth factor I (IGF-I) and IGF binding protein 3 (IGFBP-3) have also become essential laboratory tools in the diagnosis and management of GH-secretion disorders. These two analytes are secreted at high concentrations from the liver under the control of GH; they are mediators of the growth-promoting actions of GH; and they exhibit no problematic diurnal variation. The three assays thus represent a natural partnership. Within the domain of growth disorders, assays for IGF-I and IGFBP-3 have a somewhat broader application than GH assays, being important, not only for diagnosing GH deficiency (GHD), but also for monitoring therapies with recombinant human GH (rhGH) in various conditions.

The IGF system is a complex entity involving two IGFs ("somatomedins") and six IGFBPs, as well as proteases—the best known being prostate-specific antigen (PSA)—and other components.¹ From the clinical laboratory's point of view, IGF-I is the more important somatomedin because circulating levels of IGF-II are less closely regulated by GH; and IGFBP-3 stands out, not only for circulating at far higher concentrations than the other five IGFBPs, but also for its greater GH-dependence.

Ordinarily, GH hyper- and hyposecretion tend to induce corresponding increases and decreases in the circulating levels of both IGF-I and IGFBP-3, as hepatic production of both molecules is stimulated by the action of GH on its receptors. Thus, the inherent relationship between the two poles of the GH-IGF axis is one of parallelism.

A dramatically different relationship is seen, however, in the presence of GH receptor defects (e.g., Laron syndrome), which are quite rare.² Children born with such a defect exhibit severely impaired linear growth even though GH circulates at normal to elevated levels: IGF-I levels, on the other hand, remain decidedly low, as in classic GHD, often below the 0.1 centile expected for normals of comparable age.³

Growth hormone excess
Acromegaly, the most common disorder of GH hypersecretion, is often caused by pituitary tumors. In addition to treatment by conventional means (e.g., surgery, radiation, somatostatin analogs), acromegaly is commonly treated in some countries with GH receptor antagonists: this aims at blunting the usual impact of elevated GH levels rather than eliminating GH hypersecretion as such.⁴ Accordingly, GH measurements could be of no obvious help in monitoring or optimizing this treatment modality, whereas it would still make sense to look for normalization of circulating IGF-I levels.

In the diagnosis of acromegaly, however, and when this disorder is treated by conventional means, consensus guidelines from international workshops held in 1999 and 2000 specify roles for GH as well as IGF-I measurements. An IGF-I level in the normal range can help to exclude the diagnosis and is an important target for therapy. As for GH, the guidelines call for sequential measurements: either a 24-hour monitoring of spontaneous levels yielding an average GH concentration below 2.5 µg/L, or else a seemingly more cost-effective 2-hour oral glucose tolerance test (OGTT) yielding a GH result of 1 µg/L or less.⁵

But what if the GH and IGF-I results conflict? An article published in 2002 by Dimaraki et al. includes instructive data from a referral center in Ann Arbor, Michigan.⁶ Figure 1 shows a pair of 24-hour GH profiles for two adults from this study: one a normal control, the other a patient with active acromegaly confirmed by laboratory-independent criteria and as yet untreated. Although both subjects have average GH levels within (conventional) normal limits for this parameter, the acromegaly patient's IGF-I level exceeds the stated upper reference limit for the assay used.⁷

The GH profiles in Figure 1 exhibit strikingly different topographies: huge swings between peak and valley, a reflection of normal, intermittent GH secretion, versus a plateau, indicative of the persistent, "tonic" secretion characteristic of acromegaly. Statistics like the mean (or median or geometric mean) fail to discriminate between the normal and acromegaly profiles; instead, it is the lowest GH concentrations which encapsulate the relevant difference.

Figure 1. 24-hour GH secretion profiles, sampled at 10-minute intervals, for two adults. Acromegaly patient, untreated, IGF-I: 345 µg/L (elevated), GH mean: 0.6 µg/L. Normal control, IGF-I: 113 µg/L (normal), GH mean: 0.7 µg/L. Data from Dimaraki EV, et al. J Clin Endocrinol Metab 2002;87:3537-42.

Herein we see one rationale for the OGTT, as a relatively convenient way to estimate the very lowest levels expected from a 24-hour profile—rendered less precise, of course, due to sparse sampling. (From another, purely qualitative perspective, the point of an OGTT is that failure to suppress soon after a glucose load suggests an autonomous source of GH production.)

The examples published by Dimaraki et al. convey several lessons. First, for reliable measurement of the most significant levels encountered in an OGTT or spontaneous profile, one needs a GH assay with second- or third-generation sensitivity, comparable to that of the IMMULITE^® and IMMULITE^® 2000 Growth Hormone assays.

Second, for diagnosis (though not necessarily in treatment follow-up⁸), an OGTT cutoff of 1 µg/L, as recommended in the consensus guidelines, could well be set too high. A value of 0.3 µg/L—closer, presumably, to the upper reference limit for the lowest OGTT-induced GH levels in normal subjects—may prove more appropriate for today's nonisotopic, immunometric assays. Due partly to the specificity of their antibody pairs, these assays tend to yield numerically lower GH results than RIAs and first-generation IRMAs on very low patient samples.

Third, in the diagnosis and follow-up of acromegaly, an elevated IGF-I result—which can be obtained and checked far more readily than the lowest result of an OGTT—should not be ignored.

Growth hormone deficiency
In the diagnosis of GHD, where low concentrations of IGF-I and IGFBP-3 would be expected on the basis of GH hyposecretion, the interpretation of IGF-I and IGFBP-3 results is complicated by factors competing with GH secretion for control—most notably nutritional status—and by nonhepatic IGF production. Immune system status,⁹ cancers, and even social factors represent additional complications.

Malnutrition may result in low circulating levels of IGF-I, in spite of normal somatotropic function;¹⁰ while obesity may succeed in masking a case of GHD by driving IGF-I levels higher up into the normal range. It must be said that nutritional status also affects GH levels, but in opposite directions: GH secretion is typically decreased in obesity,¹¹ while fasting increases the frequency and amplitude of GH bursts.

A child with Prader-Willi syndrome (PWS), a chromosomal disorder often associated with short stature as well as a tendency (beginning at 1 to 4 years of age) towards rapid and extreme weight gain. Is GHD an inherent component of PWS? With nutritional status so often a confounding factor, it has been difficult to reach a consensus. In the United States, rhGH was recently approved for use in children with this syndrome, thus obviating the need to demonstrate GHD on an individual basis before instituting therapy. Photograph used with permission of the Prader-Willi Syndrome Association (UK), www.pwsa-uk.demon.co.uk.

IGFs act both as hormones and as cytokines: they are produced not only in the liver but at many sites throughout the body. Whereas, in general, one cannot expect local (i.e., tissue-related) changes in the distribution of IGF and IGFBP levels to be clearly discernible at the systemic level (i.e., in the circulation), it is still possible that local production might spill over into the vascular space just enough to confound the interpretation of low IGF levels.

Nevertheless, in practice, measurements of IGF-I and IGFBP-3 represent an important step in the work-up of suspected GHD, especially in children, where it is natural to evaluate these analytes before embarking on more costly studies involving sequential GH determinations. The latter remain essential to the diagnosis, however; and in spite of renewed advocacy for overnight or even 24-hour secretion profiles in children,¹² most endocrinologists have traditionally sought to document GHD with impaired responses in two different GH-stimulation tests.¹³ (Arguably, the scientific justification for these nonphysiological provocation tests lies in their ability to yield an index to the peak levels expected from a spontaneous profile.)

In adults, it is standard practice to monitor rhGH therapy with periodic IGF-I and IGFBP-3 determinations. For adult GHD, the prevailing philosophy is one of replacement; hence the desire to achieve and maintain normal circulating levels of these analytes, as surrogates for the systemic action of GH, both to minimize side-effects and because long-term exposure to high GH levels may be a risk factor for cardiovascular disease and certain epithelial cancers.¹⁴ Epidemiological studies have suggested an increased risk for such cancers in subjects with both high circulating levels of IGF-I and low circulating levels of IGFBP-3 (hence also an elevated ratio of these two analytes).¹⁵

Moreover, with the gradual appreciation that the optimal rhGH dose depends on age, gender, steroid levels and other factors, it has become increasingly common to individualize therapy in adults by starting with a low dose and titrating upwards, using IGF-I and IGFBP-3 levels for guidance.¹⁶

In children, these two analytes have also been monitored routinely "for assurance of compliance and safety," but not, until recently, for dose adjustment: generally speaking, the rhGH dose has been determined solely on weight (or surface area), as if age, gender and pubertal status were irrelevant.¹⁷

In contrast to the situation for adults, parameters related to a child's height (especially height velocity) provide an objective basis for measuring the success of rhGH administration in promoting growth. Furthermore, the list of indications for rhGH now includes several—e.g., Turner syndrome, SGA (small for gestational age), and chronic renal failure—where impaired growth remains at issue, but GHD is not assumed and need not be demonstrated.¹⁸ Accordingly, the focus of clinical research has shifted towards ways of individualizing the rhGH dose, using regression models based on laboratory results, growth-related parameters and other variables, to achieve optimal improvements in growth.

Peak levels from overnight or 24-hour spontaneous GH profiles have so far proved to be the most important of the laboratory variables in this context, but IGF-I and IGFBP-3 levels obtained prior to therapy significantly extend the scope of models for predicting first-year height velocity.¹⁹ Whether serial measurements from routine monitoring with these two analytes will also contribute towards effective dose adjustment remains an important open question.

Reference ranges
The within-day stability of circulating IGF-I and IGFBP-3 levels is a major, though hardly decisive, consideration favoring assays for these analytes over assays for GH. Published studies have demonstrated essentially no diurnal variation for IGF-I and IGFBP-3, except during rhGH therapy, which induces a modest and predictable circadian rhythm.²⁰ (See Figure 2. The difference in IGF-I levels between the two groups may be due to duration of rhGH treatment, but is more likely due to age; in any case, the two groups show a comparable degree of within-day variation.)

Figure 2. Average 24-hour IGF-I profiles, sampled at 60-minute intervals, for adults undergoing rhGH therapy. Six patients, 28–52y, after 1 week of treatment. Five patients, 50–67y, treated for 13–40 months. The lavender mound suggests the typical time course for rise and fall of circulating GH in these patients, with rhGH administered at 8 pm. Data from Oscarsson J, et al. Clin Endocrinol 1997;46:63-8.

This has immediate implications, not only for the comparative number of measurements required for reliable assessment of GH versus IGF-I and IGFBP-3, but also for the possibility of obtaining definitive interpretative frames of reference for these analytes. To establish normal values for IGF-I and IGFBP-3, only a single "random" determination is required for each subject in the reference group, whereas this is not true for GH, due to the variety of testing formats, as well as the need for multiple determinations.

Taking not just the agents, but also dosing criteria, timing, patient preparation and other significant features into consideration, GH provocation tests have been applied in an astounding number of combinations. There seems little hope for standardization; and even less for the rigorous, scientific determination of assay-specific reference values and cutoffs. It is difficult to imagine an internal review board approving normal range studies of a statistically adequate size for GH provocation tests, especially in children, given that the tests are nonphysiological and often involve some risk.

Accordingly, except possibly for overnight studies of spontaneous GH secretion—which are not open to this objection, though they are similarly arduous and costly—one must expect that cutoffs for GH tests will continue to rely on "traditional" values or on studies with very limited N-size that fail to take potentially significant variables into account—particularly age, gender, and pubertal status (Tanner stage) or steroid levels.

The same variables are relevant, of course, to IGF-I and IGFBP-3 as well. This means that establishing reference values entails a complex, multivariate study. Moreover, in this domain, it is expected that such a study will conform to the rigor and methodology associated with the development of growth standards for weight, height, height velocity and related parameters, rather than the less demanding IFCC/NCCLS guidelines for minimalist, two-point characterizations of reference distributions (e.g., in terms of central 95% reference intervals) which are still accepted as adequate in other parts of clinical chemistry.²¹

In developing reference values for IGF-I and IGFBP-3, it is also important to determine their ratio, which yields a valuable cross-check on the integrity of the data. In many applications, it is common practice to measure IGFBP-3 along with IGF-I for the same reason, that is, to use the former as an "internal control" for the latter. Furthermore—analogous to the use of T4/TBG and testosterone/SHBG ratios in other contexts—the IGF-I / IGFBP-3 ratio serves as an index of circulating free IGF-I.²²

Some of the analyses of reference values essential to the routine evaluation of growth disorders are illustrated in the accompanying figures, which show the expected distribution of IGF-I and IGFBP-3 and their ratio as a function of age (Figure 3) and Tanner Stage (Figure 4), obtained in a major recent study of children and adults in good health and of normal height.²³

Conclusions
While there are other promising applications for circulating GH, IGF-I and IGFBP-3 measurements, the best established relate to disorders of growth.²⁴ Laboratory results on their own cannot settle the issues which arise for endocrinologists, who must take far more than immunoassay results into consideration. But the laboratory's contribution can be optimized by ensuring that the assays used for these three analytes meet certain basic criteria.

For GH, a highly sensitive, specific immunometric assay is essential: automation and small sample volume are desirable features given that sequential determinations (based on provocation tests and/or spontaneous profiles) are the norm.

For IGF-I and IGFBP-3, and for their ratio, a principal requirement is rigorous, detailed, assay-specific characterizations of the normal reference distribution as a function of age, sex (where appropriate) and Tanner stage.

Figure 3. Reference range study for IMMULITE® IGF-I and IMMULITE IGFBP-3 based on ~1500 subjects in good health and of normal height. The graphs show the +2, 0, –2 and –3 SD contours estimated globally for the subjects 0.5 to 88 years of age, using modern local regression methods. Data from Elmlinger MW.

Figure 4. Tanner stage plots for 226 girls and 194 boys from the reference range study for IMMULITE® IGF-I and IMMULITE IGFBP-3. For each stage (I through V), individual results for the male (M) and female (F) subjects are displayed separately, on either side of box-and-fishbone plots for the combined data. (The box encloses 50%; vertebra pairs bracket the central 80%, 90% and 95%.) Data from Elmlinger MW.

Notes

1. IGFs, IGFBPs, proteases: [Ran97], [Juu98], [Fer99], [Gri00], [LeR01], [Fir02]. See also [R&C02], an excellent general reference, in Sperling's Pediatric Endocrinology.

2. Laron syndrome, GH resistance: [Lar99], [Lop00], [Ros01]. GHD as a subtype of IGFD: [Ros94b], [Ros96], [R&C02]. The Ecuador story: [Ros90], [Ros94a].

3. The 0.1 centile for IGF-I: [Blu94], [Blu00].

4. GH receptor antagonists: [Kop02], [Tra02a].

5. Consensus guidelines, acromegaly: [Giu00], [Mel02b]. Note that the 2002 guidelines do not mention spontaneous GH profiles. See also [Mel99], [Mel02a], [Giu03].

6. Dimaraki study, Figure 1: [Dim02]. See notes 6 and 7.

7. Apropos of [Dim02]: [Tra02b], [Fre02], [Giu03], [Vie03].

8. Not necessarily in treatment follow-up: [Cos02].

9. Immune system status: [Blu00].

10. Nutritional status: [Thi94], [Bar02], [Doh02], [R&C02], [Hol03]. Monitoring refeeding: [Cle85].

11. Prader-Willi syndrome, obesity: [Eih98], [Eih00], [Eih01], [Bar02], [Car02].

12. Spontaneous GH profiles, pro and con: [Kri01]; [Guy99], [Rog03]. See also note 19.

13. GH provocation testing. [Juu97b], [GRS98], [Sag98], [Sha98], [GRS00], [Siz01], [Jor03]. Clinical practice audits: [Wya95], [Juu02a].

14. Monitoring rhGH therapy in adults: [Juu99a], [GRS01], [Dra01], [Mur02].

15. Cancer risk: [Gri00], [Coh01], [GRS01], [R&C02], [Bar02], [Zha03]. See especially: [Coh00]. Cancer diagnosis: [Cut99], [Sta01], [Ism02], [Sco03].

16. Individualizing rhGH therapy in adults: [Dra98], [Mur00], [Van01], [Mol02], [Mur02], [Sim02].

17. Monitoring rhGH therapy in children: [Dra98], [GRS01], [Lee01], [Ran01], [Juu02a], [Spe02].

18. Use of rhGH for conditions other than confirmed GHD: [AAP97], [AACE98], [Guy99], [Juu99b], [R&C02], [Ran03].

19. Individualizing rhGH therapy in children: [Wet00], [Lee01], [Ber02], [Coh02]. Prediction models: [Alb00], [Kri01], [Ran01], [Sch01], [Kri02], [Ran03].

20. Diurnal variation: [Dra01], [Gel99], [Heu99], [Juu99a]. Figure 2: [Osc97].

21. Growth standards, age-related reference ranges: [Ran00], [Sib00], [Juu01], [Lof01], [R&C02], [Elm03].

22. IGF-I/IGFBP-3 ratio and "free" IGF-I: [Juu97a] [Heu99], [Blu00], [Ban01], [Fry01]. See also note 15.

23. Tübingen IGF-I reference range study, Figures 3 and 4: [Wen02], [Elm03], [Kuh03].

24. On the horizon: [Geu98], [Ros99], [Bar00], [Ses00], [Bar02], [Bje02], [Fry02], [Juu02b], [Daw03], [Gio03], [Hol03].

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PubMed "unique identifiers" (89 UI):

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