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A Simple Test for Growth Hormone Deficiency in Adults

University of Bristol, Division of Medicine, Bristol Royal Infirmary, Bristol BS2 8HW, United Kingdom

Address correspondence and requests for reprints to: Stafford Lightman, Division of Medicine, Dorothy Crowfoot Hodgkin Laboratories, Bristol Royal Infirmary, Marlborough Street, BS2 8HW Bristol, United Kingdom.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
GH deficiency (GHD) in adults is a well recognized clinical syndrome that results in significant metabolic and psychological morbidity. GH replacement therapy not only reverses these changes but improves the quality of life and results in a significant improvement in well being.

There is no single simple and safe test to assess GHD. GHD in adults is diagnosed biochemically by provocative testing of GH secretion, and the insulin tolerance test (ITT) is accepted to be the test of choice. However, the ITT has many contraindications, needs multiple blood samples, and is potentially dangerous, requiring regular monitoring of patients in a specialized investigation unit.

The aim of our study was to evaluate the GH-releasing effect of a combination of the hypothalamic secretagogue GHRH with a small dose of the synthetic peptide GHRP-2, to diagnose GHD. We have compared the GH response to ITT and GHRH/GHRP in a large group of adults with hypothalamic/pituitary disease (n = 36; 22 males and 14 females; age, 18–59 yr) and in healthy volunteers (n = 30; 15 males and 15 females; age, 22–66 yr).

The GHRH/GHRP test produces a measurable GH secretory response in normal, hypopituitary and GH-deficient patients. The test has no side effects. Using the ITT as our ‘gold standard’ with a GH response of 9 mU/L as our cut-off to define GHD, we compared the clinical efficacy of these two tests. Choosing an arbitrary cut-off of 17 mU/L to define GHD in the GHRH/GHRP test, this new test proved to have 78.6% sensitivity and 100% specificity even when we only used the 30-min datum point. .

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
GH DEFICIENCY (GHD) is no longer simply a pediatric condition associated with poor growth velocity. It is now becoming clear that inadequate GH secretion in adults can result in significant metabolic and psychological morbidity. Adults with GHD have reduced lean body mass, increased abdominal fat, derangements of carbohydrate and lipoprotein metabolism, impaired cardiac function, reduced exercise capacity, and reduced bone mineral content (1, 2, 3, 4, 5, 6). These changes can be reversed by replacement treatment with synthetic GH (2, 3, 4, 5, 7), and many of the subjects treated also reported a very significant improvement in well being (8). In the longer term, we know that hypopituitarism is associated with a shortened life expectancy due to increased cardiovascular mortality, although we do not yet have the data to resolve whether this can be reversed by GH replacement (7, 9).

The very large numbers of recent publications on tests of GH secretion reflect the great difficulty there has been in establishing a simple and safe test to assess GHD. The iv insulin tolerance test (ITT) is considered the ‘gold standard’ test for the diagnosis of GHD. This test, however, is potentially dangerous and entails a prolonged time in an appropriately staffed investigation unit. This makes it very expensive and inappropriate as a screening procedure outside very specialized centers. In view of this, people have assessed the value of basal measurements of IGF-I (10, 11), BP-3, and various dynamic tests or combinations of tests using GHRH, clonidine, and pyridostigmine (12, 13, 14). Unfortunately, none of these has proved satisfactory although some researchers have shown that the GHRH + arginine test was, at least, as sensitive as the ITT, provided appropriate cut-off limits were considered (17). We have now investigated a novel test in which we evaluated the GH-releasing effect of a combination of the hypothalamic secretagogue GHRH with a small dose of the synthetic GH-releasing peptide (GHRP) GHRP-2. This test not only compares very favorably with the results of the ITT, but the time course of the response is much more predictable, and a single sample taken at 30 min is adequate to differentiate GHD patients.

	Subjects and Methods

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Thirty-six adults with hypothalamic/pituitary disease (n = 36; 22 males and 14 females; age, 18–59 yr) and 30 healthy volunteers (n = 30; 15 males and 15 females; age, 22–60 yr) were studied. Details of the patients are shown in Table 1.

The study protocol was approved by the Ethics Committee of the United Bristol Healthcare Trust, and all subjects gave their informed consent. Only patients in whom there was no contra-indication to the ITT were recruited for the study. All subjects underwent, in randomized order, both an ITT and a GHRH/GHRP test. These were performed in the morning, after an overnight fast, at least 1 week apart.

Tests

ITT. Actrapid insulin (0.15 U/Kg; Novo Nordisk, Crawley, UK) was given iv at 0 min. Blood samples were taken at 0, 30, 45, 60, and 90 min. Plasma glucose fell below 2.2 mmol/L in all subjects.

GHRH/GHRP test. GHRH (1 µg/Kg; Ferring) iv and 0.1 µg/Kg GHRP-2 (courtesy of Professor C. Y. Bowers, Tulane University, New Orleans, LA) iv were both given together at 0 min. Blood samples were taken at 0, 30, 45, 60, and 90 min.

Blood samples were spun and stored at -20C. Serum GH levels were assayed by immunoradiometric assay (NETRIA IRMA). All samples from each subject were analyzed together. The sensitivity of the method was 0.2 mU/L.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Healthy volunteers

There was a very prompt release of GH in response to GHRH/GHRP (Fig. 1). Peak response (mean, 110.9 mU/L; range, 19–240) occurred at 30 min in 29 of the 30 volunteers and at 45 min in the remaining subject. In contrast, the peak GH response during ITT (mean, 59.9 mU/L; range, 14.4–155.5) occurred at the varied times; 0 of 30 at 30 min, 2 of 30 at 45 min, 11 of 30 at 60 min, and 17 of 30 at 90 min.

View larger version (18K): [in this window] [in a new window]   Figure 1. The GH response to the insulin tolerance test (-•-) and the GHRH/GHRP test (--) in normal control subjects and hypopituitary subjects who had been defined as either GH deficient or non-GH deficient based on their GH response to hypoglycemia of < or > 9 mU/L.

Patients with hypothalamic/pituitary disease

There was also a very prompt release to GHRH/GHRP in these patients (Fig. 1). Peak response occurred at 30 min in 29 of 36 patients, at 45 min in 5 of 36 patients, and at 60 min in the remaining 2 patients. Again, the GH response during ITT occurred at much more variable times: 4 of 36 at 30 min, 5 of 36 at 45 min, 12 of 36 at 60 min, and 15 of 36 at 90 min. There were no side effects from GHRH/GHRP in all three groups.

Comparison of the two tests

There was a strong correlation between the GH response to the ITT and to the GHRH/GHRP test (r = 0.715, P < 0.001, Pearson correlation test).

There is no absolute definition for the criteria of severe GHD. Recent consensus guidelines define severe GHD as a peak GH response to hypoglycemia of less than 9 mU/L, provided the GH assays use polyclonal competitive RIAs (15, 16) calibrated against a specific pituitary-derived preparation IRP 80/505. Using this criterion as the basis for our diagnosis of severe GHD (peak GH 9 mU/L), 28 of our 36 patients were severely GH deficient (Fig. 2). The other eight patients had a mean peak GH response of 35.5 mU/L (range, 14.6–77.1). Using the ITT data to classify our patients, we grouped our GHRH/GHRP results according to the ITT diagnosis. On this basis, the GHD patients showed a mean peak GH response of 10.0 mU/L (range, 0.7–34.9) and the other patients showed a mean peak GH response of 74.05 mU/L (range, 19.3–189.5) to the GHRH/GHRP test.

View larger version (20K): [in this window] [in a new window]   Figure 2. GH levels (mU/L) in healthy volunteers (A, D, and G) and in patients with hypothalamic or pituitary disease who are defined as GH deficient (B, E, and H) or not GH deficient (C, F, and I) based on their GH response to hypoglycemia of < or > 9 mU/L. These data show peak levels of GH during the ITT () (A–C), the GHRH/GHRP test () (D–F), as well as the GH level at 30 min after the GHRH/GHRP test () (G–I). Dotted lines for columns A–C represent 9 mU/L GH, and in D–I represent 17 mU/L GH. The six patients who showed suboptimal responses to ITT (•), but were GH sufficient during the GHRH/GHRP test (), are included in groups B and F, respectively. Their GH levels at 30 min after the GHRH/GHRP test are shown in group I ().

Using our criteria for severe GHD (peak GH <17 mU/L) during the GHRH/GHRP test, 22 of our 36 patients were found to have severe GHD. Six patients who were found to have subnormal GH responses during the ITT (peak GH 9 mU/L) had normal GH responses to the GHRH/GHRP test. Two of these patients had childhood onset GHD and were of short stature, suggesting hypothalamic GHD. There is already good evidence that the GHRH/GHRP test evokes a better GH response in patients with ‘hypothalamic’ GHD as compared with the ITT (22, 23). The remaining four patients (two with macroprolactinomas and two with endocrinologically inactive pituitary macroadenomas) had coexistent hyperprolactinemia with PRL levels ranging from 400–1000 mU/L. The coexisting hyperprolactinemia could well explain the insufficient GH peak during the ITT, contrasting with a normal response to the GHRH/GHRP test. This has been well documented and has been attributed to various mechanisms, which we have elaborated on in our discussion (24). If we exclude these six patients, all patients diagnosed as GH deficient by ITT criteria had a peak GHRH/GHRP response of <16.7 mU/L whereas all patients with a response of GH of >9 mU/L during ITT had a peak GHRH/GHRP response of >18 mU/L (Fig. 2).

If instead of looking for peak response to GHRH/GHRP we only look at the result at 30 min and define severe GHD with the same criteria as that used for peak responses to GHRH/GHRP (cut off at 17 mU/L), there is 100% concurrence in diagnosis for all our subjects. If, on the other hand, the ITT is considered to be the reference test, then the GHRH/GHRP test has 78.6% sensitivity and 100% specificity even when the 30-min datum point alone is used.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
There is a clear need for a simple and safe test to establish whether a patient has a deficiency of GH secretion. In addition to the ITT, there are a variety of stimulation tests including GHRH, glucagon, arginine, clonidine, and pyridostigmine, or combinations of these agents. The ITT test has potential side effects and demands experienced staff and multiple samples. The other tests have a variety of defects, mainly considerable inter subject variability in GH response, but also a high rate of false positive results. They also need multiple samples to be taken over a period of hours.

The GHRH/GHRP test uses the endogenous GH secretagogue GHRH and a synthetic peptide GHRP-2, which is a relatively selective GH secretagogue acting directly at both hypothalamic and pituitary levels (18, 19, 20, 21). GHRP has a synergistic effect with GHRH on GH release (19). Although the endogenous ligand is unknown, a receptor has been cloned, and in situ hybridization has confirmed its presence in the arcuate nucleus of the hypothalamus and in the anterior pituitary (21).

Our study compares the peak GH response to the ITT and GHRH/GHRP tests and demonstrates a higher peak GH response to the GHRH/GHRP test as compared with the ITT in healthy volunteers, as well as patients with hypothalamic/pituitary disease. We also recognize that the modest agreement between peak GH levels in response to the ITT and the GHRH/GHRP test is explained by a poor reproducibility of GH stimulation tests and by the different mechanisms of action of the stimuli used.

The higher peak GH responses to GHRH/GHRP as compared with the ITT in four patients with hyperprolactinemia are probably explained by hypoglycemia and GHRH/GHRP having different stimulatory effect on the pituitary somatotroph. PRL secretion is predominantly under negative dopaminergic control, and an elevated PRL in pituitary adenomas is interpreted as a sign of functional hypothalamic-pituitary interruption (25). Hyperprolactinemia does not affect the peak GH response to ITT, suggesting an unchanged GHRH tone in hyperprolactinemia. Given the complexities of hypothalamic-pituitary GH regulation, there is no doubt that additional hormonal mechanisms play an important role in hypopituitarism, hyperprolactinemia, and altered pituitary GH responsiveness. Our study confirms previous findings with GHRH alone (24) that hyperprolactinemia is associated with higher GHRH-stimulated GH without having an effect on the GH response to ITT.

Previous studies on the GH-releasing effects of GHRH/GHRP-6 combinations have shown GH stimulation in excess of 10 mU/L in adult patients with long-standing GHD and impaired GH responses to ITT (22), and a poor response both in patients with GHD and stalk compression (23). Detailed dose-response studies on the effect of GHRP-2 both in the presence and in the absence of GHRH suggest that low-dose GHRP-2 are more relevant to the physiological regulation of GH release (26). Based on these data, we chose a low dose of GHRP-2 that also avoids a ceiling effect of giving supramaximal doses of both agents. At the doses we used we found a good reproducible GH secretory response in normal males and females within the age range of 22–66 yr. Furthermore, the rapid response ensures that a single sample taken at 30 min is adequate to define responsiveness of the GH axis. The test has no detectable side effects. We suggest that the GHRH/GHRP test should be used as a very simple way to detect GHD in the outpatient department.

Received June 25, 1999.

Revised August 23, 1999.

Accepted January 6, 2000.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

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