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Endocrine Care |
Institute of Endocrinology, Diabetes and Diseases of Metabolism, University Clinical Center (V.P., S.P., M.D.), Institute of Radiology and Oncology (I.G.), 11000 Belgrade, Yugoslavia; and Department of Physiology (C.D.) and Medicine (F.F.C.), Endocrine Division, Santiago de Compostela University, E15780 Santiago de Compostela, Spain
Address all correspondence and requests for reprints to: Prof. Dr. Vera Popovic, Institute Endocrinology, Diabetes mellitus and Metabolism, University Clinical Center, Dr. Subotic 13, 11000 Belgrade, Yugoslavia. E-mail: . popver{at}eunet.yu
Abstract
Patients treated with cranial radiation are at risk of GH deficiency (GHD). We evaluated somatotroph responsiveness to maximal provocative tests exploring the GH releasable pool in relation to the impact of radiation damage to the hypothalamic-pituitary unit. The GH-releasing effect of GHRH plus GH secretagogue [GH-releasing peptide (GHRP)-6] (GHRH+GHRP-6) was studied in 22 adult patients (age, 23.2 ± 1.4 yr; 8 female and 14 male; mean body mass index, 22.6 ± 0.7 kg/m2) who received cranial radiation for primary brain tumor distant from hypothalamic-pituitary region 7.6 ± 0.7 yr before GH testing. Two stimulatory tests for GH secretion were employed: insulin tolerance test (ITT, 0.15 IU/kg regular insulin iv bolus); and GHRH+GHRP-6 test: GHRH (Geref Serono, Madrid, Spain; l µg/kg) plus GHRP-6 (CLINALFA, Laufelingen, Switzerland; 1 µg/kg) as iv bolus. Serum GH was measured (Delphia; Perkin Elmer, Wallac, Turku, Finland) at -30, -15, 0, 15, 30, 45, 60, 90, and 120 min. Anterior pituitary function was normal in all except in 1 female with hyperprolactinemia. Twelve out of 22 irradiated patients were GH-deficient (GHD) with both tests. Eleven out of 22 (50%) irradiated patients were severely GHD, according to the ITT (GH < 3 µg/liter; mean GH peak, 1.5 ± 0.5 µg/liter). In 9 irradiated patients, in whom ITT was performed as well, mean peak GH after the GHRH+GHRP-6 test was 6.2 ± 0.8 µg/liter, which is considered as severe GHD, according to our own cut-off for the test (peak GH < 10 µg/liter). GH responses to both tests were highly concordant, but the differential in the GH peak concentrations between GHD and non-GHD irradiated patients was significantly larger for the GHRH+GHRP-6 test than that for the ITT. The 2 discordant responses, i.e. poor response to the ITT and good response to the GHRH+GHRP-6 test, were found in 1 hyperprolactinemic female patient and in 1 other female. One irradiated patient was diagnosed as GHD only with the combined test, because ITT was contraindicated because of epilepsy. PRL and cortisol responses to ITT were normal in all irradiated patients and did not depend on the GH status. IGF-I levels were not informative or discriminative between the GHD and non-GHD irradiated adult patients. In conclusion, the use of GH secretagogues plus GHRH is an easy, reliable and accurate way of assessing GH secretion in cranially irradiated patients. Impairment of the GH releasable pool in the irradiated patients, with a maximal provocative test, reflects alterations in the hypothalamic-pituitary unit caused by radiotherapy.
THE GRADUAL DEVELOPMENT of unnatural GH-releasing peptides (GHRPs) has led to the proposal of new GH provocative tests (1). We have recently proposed that administering iv bolus GHRH+GHRP-6 causes a higher peak GH response, as compared with the insulin tolerance test (ITT), in healthy volunteers as well as in patients with hypothalamic/pituitary disease and that the GH response is rapid (within 30–45 min) (2). The test has high sensitivity and specificity. The combined GHRH+GHRP-6 test is simple and reproducible and has greater safety and ease than the current so-called gold-standard ITT in diagnosing GH deficiency (GHD) in adults. We have also shown that the GHRH+GHRP-6 test is not affected by mild physical activity and meals (3).
The synergistic action of combined peptides (GHRH+GHRP-6) on GH release in vivo is not explained by the direct pituitary action. A more global action on the hypothalamic-pituitary unit is proposed because GHRPs act via specific G protein-coupled receptor expressed in the pituitary gland and in several hypothalamic nuclei (4, 5). A problem that arises in the use of the GHRH+GHRP-6 in the diagnostic approach for GHD of hypothalamic origin concerns the unknowns that still exist at both the approach and clinical disorder levels (6).
Cranial irradiation may lead to hypothalamic and pituitary dysfunction (7, 8). The pathophysiology of the radiation-induced damage to the hypothalamus remains ill understood, and it is thought that direct injury to hypothalamic neurons, rather than reduced cerebral flow, is the major cause of progressive hypothalamic-pituitary dysfunction after fractionated cranial irradiation (9). It was suggested that GHRH+GHRP-6, the combined test, should be used for investigation of adults with suspected GHD in patients with organic pituitary disease, whereas the ITT remains the preferred test in patients with hypothalamic disease because it stimulates GH release indirectly through the hypothalamus (8, 10). Therefore, there is a possibility that combined GHRH+GHRP-6 testing could be susceptible to false negative testing by inducing GH release in patients with a hypothalamic cause of GHD. The study assesses the reliability of the combined test in a cohort of cranial irradiated patients who are likely to have radiation damage to the somatotropic axis.
Subjects and Methods
Subjects
We studied 22 adult patients, 23.2 ± 1.4 yr old (range, 16–41 yr), of whom 8 were female and 14 male [mean body mass index (BMI), 22.6 ± 0.7 kg/m2], who received cranial radiation (XRT) for primary brain tumors distant from the hypothalamic-pituitary axis 7.6 ± 0.7 yr before provocative testing (range, 2–13 yr). The age of the patients at diagnosis and cranial radiation was 15.6 ± 1.3 yr (range, 6–30 yr). The tumor diagnoses were medulloblastoma (n = 12), tumors of the pineal region (n = 4), ganglioglioma (n = 3), meningioma anaplasticum (n = 1), and ependymoma (n = 2). Patients received, after surgical treatment, a mean radiation dose of 52.8 ± 1.4 Gray (Gy) directed at the primary tumor, whereas the estimated dose delivered to the hypothalamus/pituitary ranged from 25–30 Gy. Three patients received spinal irradiation in addition to cranial irradiation, at prepubertal age (6, 7, and 9 yr). In the periodic follow-ups, they were considered free of the initial disease. The patients’ characteristics are summarized in Table 1
. Anterior pituitary function (thyroid, gonadal, hypothalamo-pituitary-adrenal axis) was assessed in all patients. All but 2 patients reached normal adult height because of the irradiation of the craniocervical region. None of our patients received GH in the past.
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Provocative tests
Each irradiated patient and control underwent two stimulatory tests of GH secretion on different days, separated at least by 1 wk: 1) insulin-induced hypoglycemia (ITT) with 0.15 U/kg regular insulin iv (iv at time 0 min); and 2) combined GHRH+GHRP-6 test [GHRH (1 µg/kg, GRF 1–29 NH2, Geref Serono) plus GHRP-6 (1 µg/kg, His-D-Trp-Ala-Trp-D-Phe-Lys-NH2; CLINALFA)]. Blood samples were obtained at 0, 30, 60, 90, and 120 min during ITT and at -30, -15, 0, 15, 30, 45, 60, 90, and 120 min during GHRH+GHRP-6. During the ITT, satisfactory hypoglycemia was documented clinically and biochemically (blood glucose <2.2 mM). GHD was defined as a peak GH concentration less than 3 µg/liter during ITT and less than 10 µg/liter during GHRH+GHRP6. Basal serum IGF-I concentrations were also measured.
All patients were adequately tested for other anterior pituitary axes (thyroid, adrenal, gonadal, and four-point day curve for PRL).
Methods
GH levels were measured with a time-resolved fluoroimmuoassay (Wallac, Inc., Turku, Finland; µg/liter), with sensitivity of the assay being 0.011 µg/liter and with coefficient of variation (CV) values of 6.3% (0.4 µg/liter), 5.3% (10.2 µg/liter), and 4.2% (43.4 µg/liter). IGF-I was measured by RIA (Nichols Institute Diagnostics, San Juan Capistrano, CA) with an intraassay CV of 5.2% and an interassay CV of 9.4%.
All data were reported as mean values ± SEM. GH responses to provocative stimuli were quantified by determining area under the curve (AUC), calculated using the trapezoid rule. Nonparametric methods were used for statistical analysis, including the Mann-Whitney U test for independent samples, the Wilcoxon signed-rank test for paired data, and the Spearman correlation, considered significant at P value less than or equal to 0.05.
Results
Anterior pituitary function was normal in all patients except in one. This female patient had hyperprolactinemia and oligomenorrhea and was on estrogen replacement therapy. Two patients were given L-T4 (100 µg daily) replacement therapy for primary hypothyroidism (attributable to irradiation of craniocervical region).
The ITT
Eleven out of 22 (50.0%) irradiated patients were severely GH-deficient (GHD) (peak GH response <3 µg/liter, after hypoglycemia). In GHD patients, compared with non-GHD patients, the GH peak after hypoglycemia was 1.5 ± 0.5 vs. 15.9 ± 3.1 µg/liter, P < 0.001 (Fig. 1); whereas AUC-GH was 77.7 ± 20.1 vs. 948.5 ± 179.4 µg/liter·120 min, respectively (P < 0.001). There was no difference in GH response to ITT between healthy controls and non-GHD irradiated patients (P > 0.05).
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Nine irradiated patients in whom both tests were performed were diagnosed as GHD after the GHRH/GHRP-6 test, according to our own cut-off (<10 µg/liter) (2), with mean peak GH of 6.2 ± 0.8 vs. 54.8 ± 9.4 µg/liter in non-GHD irradiated patients, P < 0.001 (Figs. 1
and 2); whereas AUC-GH was 386.6 ± 192.7 µg/liter·120 min in GHD vs. 3359.4 ± 547.3 µg/liter·120 min (P < 0.001) in non-GHD patients. There was no difference in GH response to GHRH+GHRP-6 between healthy controls and non-GHD irradiated patients (P > 0.05, Fig. 2). In one patient, only the combined test was performed, because of epilepsy; and, because her GH peak concentration was 6.7 µg/liter, she was diagnosed as GHD.
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In cranially irradiated patients, the GH response to GHRH+GHRP-6 was positively associated and highly concordant with those to ITT (r = 0.456, P = 0.043, Fig. 3). Analyzing individual GH responses, only 2 persons out of 22 cranially irradiated patients had GH peaks below 3 µg/liter after ITT, whereas GH peaks after the combined test were above 20 µg/liter (1 female had hyperprolactinemia, as a sign of hypothalamic dysfunction; Fig. 4).
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GHD patients had lower IGF-I levels, compared with those of non-GHD patients (28.6 ± 3.4 vs. 40.4 ± 5.2 nM; P = 0.06), but still in the normal range (15–40 nM). There was overlap in the IGF-I values between the two groups.
Discussion
Half of our cranially irradiated patients developed GHD, estimated by either the preferred test, the ITT (10) (stimulating GH secretion indirectly through hypothalamus), or the combined test (stimulating GH release at both the hypothalamic and pituitary levels). In the majority of irradiated patients, we have shown that the responsiveness of GH to the GHRH+GHRP-6 test is severely impaired in a manner parallel to that of the ITT. GH responsiveness to both tests gave the same pattern of response, suggesting radiation damage to the whole somatotropic axis. Our present data demonstrate that a provocative test as potent as GHRH+GHRP-6 shows impairment of the GH releasable pool in cranially irradiated patients. One GHD patient, according to the combined test (GHRH+GHRP-6), was not tested with ITT because of epilepsy. Only two female patients showed discordant GH response to ITT vs. GHRH+GHRP-6, i.e. had poor response to ITT and exuberant response to the combined test. These two patients may thus confirm the previous observations that hypothalamus is more vulnerable to radiation damage than is the pituitary (9) and that only in a few cases may the combined test fail to distinguish patients with GHD from normal subjects. On the other hand, these patients would, in the future, benefit from treatment with GHRH analogues and/or GH secretagogues, thus making this test very valuable. Hyperprolactinemia found in one of these two patients confirms previous observations (11, 12) that hyperprolactinemia is associated with higher GHRH-stimulated GH, without having an effect on the GH response to ITT.
The diagnostic value of a GH provocative test depends on the prior probability of disease. The predictive value of a GH provocative test depends on the presence of organic hypothalamic-pituitary disease in the tested subject. It is estimated that the prevalence of GHD in organic pituitary disease is about 80%. Thus, the predictive value of the ITT in these patients is about 98%. On the other hand, the prevalence of GHD in the general population seems to be about 0.02%; and thus, the predictive value of the ITT in this setting is only 0.6% (13). So, although GH responsiveness to provocative agents is stimulus-dependent in irradiated patients, these individuals have high prevalence of GHD (more than half), and the predictive value of the tests should be high (7). We have previously proposed that the cut-off for normality for the GHRH/GHRP-6 test is more than 20 µg/liter and for GHD, less than 10 µg/liter (2). Every GH peak located between 20 and 10 µg/liter should be considered uncertain, but the predictive value is greater when considering the clinical information and additional hormonal deficiencies. In addition, the differential between non-GHD irradiated patients and GHD patients was much larger for the GHRH/GHRP-6 test than for the ITT, allowing a larger safe zone for interpretation.
The frequency of GHD exceeds 95% in all patients with more than one additional deficiency (14), and the likely proportion of patients with intact GH secretion in the group with a single deficient axis is only 7% (15, 16). None of our irradiated patients with GHD was in the uncertain zone, and none of them had additional pituitary hormone deficiencies, except one with hyperprolactinemia and hypogonadism. Thus, these results confirm that our previously selected diagnostic threshold (cut-off) for GHD after the combined test, of less than 10 µg/liter, is accurate.
Our study confirms once more that, in adults, the sensitivity of IGF-I in diagnosing GHD patients is low. The predictive value of a normal IGF-I in adult patients with pituitary disease is about 57%, which means that 4 patients out of 10 with GHD would have been missed (13).
In conclusion, we have shown that patients who have had cranial radiotherapy may have substantial hypothalamic-pituitary dysfunction, suggested by a blunted response to GHRH/GHRP. In only a few (16% of patients), an ITT will likely be necessary.
Acknowledgments
Footnotes
Abbreviations: AUC, Area under the curve; BMI, body mass index; CV, coefficient of variation; ITT, insulin tolerance test; GHD, GH-deficient (or GH deficiency); GHRP, GH-releasing peptide; GHRH+GHRP-6, GHRH plus GHRP-6; XRT, cranial radiation.
Received October 11, 2001.
Accepted February 6, 2002.
References
This article has been cited by other articles:
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  K. Chihara, A. Shimatsu, N. Hizuka, T. Tanaka, Y. Seino, Y. Katofor, and the KP-102 Study Group A simple diagnostic test using GH-releasing peptide-2 in adult GH deficiency Eur. J. Endocrinol., July 1, 2007; 157(1): 19 - 27. [Abstract] [Full Text] [PDF]
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 S. E. Lerner, G. J. M. Huang, D. McMahon, C. A. Sklar, and S. E. Oberfield Growth Hormone Therapy in Children after Cranial/Craniospinal Radiation Therapy: Sexually Dimorphic Outcomes J. Clin. Endocrinol. Metab., December 1, 2004; 89(12): 6100 - 6104. [Abstract] [Full Text] [PDF]
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