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Comparison of diagnostic values of growth hormone stimulation tests in adolescents/ Adolesanlarda buyume hormonu stimulasyon testlerinin tanisal degerlerinin karsilastirilmasi.

Introduction

The axis of growth hormone (GH) and insulin-like growth factor-1 (IGF-1) must be evaluated in several conditions, i.e. severe short stature (height SD score <-3 SD), severe growth deceleration (height velocity <-2 SD) or less severe short stature (height SD score between -2 and -3 SD) and growth deceleration (height velocity <-1 SD). History of brain tumor, cranial irradiation, or other organic pituitary abnormalities and radiological evidence of abnormality of the pituitary are the other conditions that must be evaluated (1).

GH is synthesized in somatotrope cells, which are present in the anterior pituitary gland. GH plays a role in normal growth and also in the regulation of carbohydrate, protein, lipid and mineral metabolisms. In addition to direct effects of GH on peripheral tissues, its indirect effects are related with IGF-1, which is produced by liver and other tissues. The secretion of GH appears in a pulsatile form; these pulses are 8 to 12 times a day (2). Between these pulses, level of GH is low. Because of this pulsatile pattern, diagnosis of GH deficiency (GHD) requires stimulation tests (1,3,4).

GH can be stimulated by physiological or pharmacological ways. Sleep, fasting and exercise are the physiologic stimuli of the GH. Pharmacological stimuli include L-dopa, clonidine, propranolol, glucagon, arginine, GH-releasing hormone (GHRH) and insulin-induced hypoglycemia. In the diagnosis of GHD, these pharmological stimuli can be used alone or in a variety of combinations. Since no one of the GH stimulation tests have 100% specificity or 100% sensitivity, at least two tests are performed (5-7). Therefore, we investigated sensitivity, specificity, positive and negative predictivities of insulin-induced hypoglycemi-a, clonidine, L-dopa and glucagon tests in subjects with severe short stature.

Materials and Methods

Patients

We evaluated forty-three patients (32 males, 11 females) with short stature admitted to the Endocrinology Department at Harran University. All patients had severe short stature with height standard deviation score less than 3 SD. The patient aged between 11 and 20 years and the mean chronological age was 14.9[+ or -]2.3. The bone age of the patients was at least two years delayed (12.4[+ or -]2.8). Median height was 139.0 (111-158), median weight was 34.0 (23.0- 58.5) and median body mass index (BMI) was 16.6 (13.3-32.5) (Table 1).

Test Procedures

L-dopa, clonidine and glucagon were applied to the patients in various combinations in addition to insulin tolerance test (ITT), which was performed to all patients. Clonidine, L-dopa and glucagon tests were applied to 21, 32 and 12 patients, respectively. Two tests were performed in 21 patients and three tests in 22 patients. Before the tests, patients fasted for a 10-hour overnight period. Then, GH stimulation tests were performed according to the following procedures at 08.30-09.00 a.m:

1) ITT: 0.10-0.15 U/kg intravenous regular human insulin was given and the target blood glucose level was less than 45 mg/dL. Glucose levels of the patients were measured every five minutes and the test was terminated when hypoglycemia occurred.

2) L-dopa test: L-dopa was given perorally accounted for by body weight; 125 mg for less than 15 kg, 250 mg for 16kg to 30 kg and 500 mg for over 30 kg.

3) Clonidine test: Clonidine was given peroral accounted for by body weight; 50 mcg for 5-15 kg, 100 mcg for 16-25 kg, 150 mcg for 26-35 kg, 200 mcg for 36-45 kg, and 250 mcg for over 46kg.

4) Glucagon test: It was administered 0.03 mg/kg (maximum 1 mg) subcutaneously.

Blood samples were collected every 15 minutes from 0 to 60 minutes and also when hypoglycemia was detected for the ITT and were taken every 30 minutes from 0 to 120 minutes for the other three tests. A value of serum GH concentration equal/over 10 ng/mL was accepted as positive response to the test.

GH Assay

Serum GH concentrations were determined in the patients by two-site chemiluminescent enzyme immunometric assay (hGH Immulite, Siemens, Llanberis, UK).

Statical Analyses

Response differences between test groups were analyzed by [chi square] test. A P value <0.05 was considered statistically significant. The results are given as mean[+ or -]standard deviation. Specificity, sensitivity, positive predictive and negative predictive values were calculated for each test using the numbers of patients with true positive (TP), true negative (TN), false positive (FP) and false negative (FN) results. In these analyses, patients with no response to two tests were classified as having GHD. Patients with GHD were classified as TP or FN according to their positive or negative responses for each tests. Sensitivity was defined as the percentage of patients with GHD, who had no response to test (calculated as TP/ITP+FN]). Specificity was defined as the percentage of the subjects without GHD, who had positive response to test (calculated as TN/[TN+FP]). Positive predictive value (PPV) was defined as the possibility of GHD in the subjects with no response to test (calculated as TP/[TP+FP]). Negative predictive value (NPV) was defined as the possibility of determining an individual without GHD among the subjects with response to test (calculated as TN/[TN+FN]).

Results

ITT was administered to all 43 patients and positive response to the test (i.e GH [greater than or equal to] 10ng/mL) was detected in 15 of them (34.6%). Hypoglycemia was occurred at a mean time of 18.7 minutes and the mean value of glucose was 38.9 mg/dL. L-dopa, clonidine and glucagon tests were administered to 32, 21 and 12 patients and positive responses to the tests were detected in 11 (34.4%), 12 (57.1%) and 2 (16.7%) patients, respectively (Table 2).

The comparisons of the L-dopa, clonidine and glucagon test results with the ITT response were not statically significant (Table 3). We diagnosed GHD in a patient, if the patient had no response to two tests. We found GHD in 23 patients. In determining GHD, the sensitivities of the ITT, clonidine and L-dopa tests were 91%, 72% and 94% respectively. Besides, the specificities of the tests were 65%, 90% and 66%, respectively. Sensitivity and specificity of the glucagon test were 100%. In addition PPV and NPV of the tests were analyzed; PPV and NPV of the glucagon test were 100%. PPV of the clonidine test was higher than of the other two tests, but NPV of the clonidine test was low. ITT and L-dopa had similar PPV and NPV. The results are shown in Table 4 and Table 5.

Discussion

In the evaluation of severe short stature, distinguishing GHD from other reasons for growth retardation is very important. Because of no gold standard test, at least two GH stimulation tests are required for the diagnosis of GHD (1,8,9). Although none of the GH stimulation tests have 100% specificity and 100% sensitivity, it has been accepted that ITT should be one of the preferred stimulation tests (6,7). The protocols of stimulation tests including arginine, ITT, clonidine, L-dopa and glucagon are well-standardized. These tests should be performed by an experienced team and monitored carefully because of their important side effects (8). Shah et al. reported two deaths and one neurological damage after GH stimulation tests (10). Binde et al. reported an-8 year-old girl who had cardiac arrest after ITT (11). In this study, we evaluated forty three patients with severe short stature by using variable combinations of the stimulation tests. We performed the tests to the patients while hospitalized and all patients were observed carefully for side effect of the tests. We did not record any severe side effect.

There is no gold standard test for the diagnosis of GHD (8,12) and there is no consensus on the cut-off level. Especially, GH values below 10 ng/mL in two GH stimulation tests are accepted as GHD in most countries (7). Tillman et al. compared GH stimulation tests with IGF-1, IGF binding protein-3 (IGFBP-3) and urinary GH excretion in the diagnosis of childhood GHD. According to this study, GH stimulation test was the most useful test to exclude GHD, when cut-off point was 10 ng/ml (13). ITT is generally accepted as the first preferred stimulation test. The advantages of this test are that range between normal and severe GHD is large, and moderate hypoglycemia achieves maximal GH release. Furthermore, ACTH-adrenal axis can be evaluated at the same time (4,6,14). We also used ITT as a first test and performed it to all patients.

Clonidine is an alpha-2 adrenergic agonist agent. Clonidine increases growth hormone-releasing hormone (GHRH) and inhibits somatostatin release. It is a safe, reliable, sensitive agent in the stimulation of GH. Most important side effects of this agent are hypotension and drowsiness (1,15,16). L-dopa stimulates releasing of GHRH via an alpha-adrenergic mechanism. Most important side effects of L-dopa are nausea, emesis and headache (1,17). Glucagon stimulates endogenous insulin and subsequently GH secretions. ITT may create a risk in newborn and small children, and therefore, glucagon seems to be a very good option for them. Nausea and vomiting may be seen as a side effect. During the glucagon stimulation test, hypoglycemia had not be reported (1,18,19).

In our study, we estimated specificity, sensitivity, PPV and NPV of each stimulation tests according to presence of GHD. The sensitivity and the specificity of the glucagon test were 100%. The sensitivities of ITT and L-dopa test were found nearly at the same level (91% and 94%, respectively). Clonidine test was found less sensitive (72%) than ITT an L-dopa tests. However, the specificity of the clonidine test (90%) was higher than those of ITT and L-dopa test (65% and 66%, respectively). Biller et al. compared five stimulation tests (ITT, L-dopa, arginine, L-dopa+arginine and GHRH+arginine) and serum insulin- like growth factor levels in adults. According to this study, specificities of ITT and L-dopa were 96% and 100%, and sensitivities of these tests were 92% and 62% (20). We found that the sensitivities of two tests were similar to this study, but specificities of the tests were lower in our study. This discrepancy may be related with selected population (adolescent population in our study). Al-Ruhaily et al. studied patients with short stature aged 12-21 years. They compared clonidine test with ITT. The sensitivity and specificity of clonidine test were 81% and 82% and those of ITT were 65% and 59%, respectively (16). We found clonidine test less sensitive but more specific than ITT.

Conceicao et al. showed that glucagon test had 88% sensitivity and 97% specificity in adults with GHD, when cut-off point was taken as 3 ng/mL. PPV and NPV of glucagon test were 100% in their study (21). Also Gomez et Al. studied glucagon test in adults and they took 3 ng/mL as a cut-off point. They showed that sensitivity and specificity of glucagon test were 100% (22). Similar to these studies, we found specificity, sensitivity, PPV and NPV of glucagon to be 100% in adolescents.

Conclusion

In conclusion, glucagon, L-dopa and ITT have high negative predictive values, therefore, these tests, especially the glucagon test, should be firstly used in the evaluation of adolescent GHD patients. After excluding the patient with adequate GH response, the test with the highest positive predictive value (i.e. clonidine) might be used to detect the subjects with GHD.

References

(1.) Richmond EJ, Rogol AD. Growth hormone deficiency in children. Pituitary 2008; 11: 115-120.

(2.) Sam S, Frohman LA. Normal Physiology of Hypothalamic Pituitary Regulation. Endocrinology Metabolism Clinics of North America 2008; 37: 1-22.

(3.) Brabant G, Rasmussen AK, Biller BMK, et al. Clinical Implications of Residual Growth Hormone (GH) Response to Provocative Testing in Adults with Severe GH Deficiency. Journal of Clinical Endocrinology and Metabolism 2007; 92: 2604-2609.

(4.) Ayuk J, Sheppard MC. Growth hormone and its disorders. Postgraduate Medical Journal 2006; 82: 24-30.

(5.) Rosenfeld RG, Albertsson-Wikland K, Cassorla F, et al. Diagnostic Controversy: The Diagnosis of Childhood Growth Hormone Deficiency Revisited. Journal of Clinical Endocrinology and Metabolism 1995; 180: 1532-1540.

(6.) Shalet SM, Toogood A, Rahim A, Brennan BMD. The Diagnosis of Growth Hormone Deficiency in Children and Adults. Endocrine Reviews 1998; 19: 203-223.

(7.) Guyda HJ. Growth Hormone Testing and the Short Child. Pediatric Research 2000; 48: 579-580.

(8.) GH Research Society. Consensus Guidelines for the Diagnosis and Treatment of Growth Hormone (GH) Deficiency in Childhood and Adolescence: Summary Statement of the GH Research Society. Journal of Clinical Endocrinology and Metabolism 2000; 85: 3990-3993.

(9.) Obara-Moszy/ska M, Kedzia A, Korman E, Niedziela M. Usefulness of growth hormone (GH) stimulation tests and IGF-I concentration measurement in GH deficiency diagnosis. Journal of Pediatric Endocrinology and Metabolism 2008; 21: 569-579.

(10.) Shah A, Stanhope R, Matthew D. Hazards of pharmacological tests of growth hormone secretion in childhood. British Medical Journal 1992; 304: 173-174.

(11.) Binder G, Bosk A, Gass M, Ranke MB, Heidemann PH. Insulin tolerance test causes hypokalaemia and can provoke cardiac arrhythmias. Hormone Research 2004; 62: 84-87.

(12.) Gandrud LM, Wilson DM. Is growth hormone stimulation testing in children still appropriate? Growth Hormone and IGF Research 2004; 4: 185-194.

(13.) Tillmann V, Buckler JMH, Kibirige MS, et al. Biochemical Tests in the Diagnosis of Childhood Growth Hormone Deficiency. Journal of Clinical Endocrinology and Metabolism 1997; 82: 531-535.

(14.) Aimaretti G, Corneli G, Razzore P, et al. Comparison between Insulin-Induced Hypoglycemia and Growth Hormone (GH)-Releasing Hormone + Arginine as Provocative Tests for the Diagnosis of GH Deficiency in Adults. Journal of Clinical Endocrinology and Metabolism 1998; 83: 1615-1618.

(15.) Lanes R, Recker B, Fort P, Lifshitz F. Low-dose oral clonidine. A simple and reliable growth hormone screening test for children. American Journal of Diseases of Children 1985; 139: 87-88.

(16.) Al-Ruhaily AD, Malabu UH. Growth hormon deficiency sort stature in a third world adult endocrine clinic. Usefulness of clonidine test in its diagnosis. Indian Journal of Medical Sciences 2008; 62: 149-156.

(17.) Collu R, Leboeuf G, Letarte J, Ducharme JR. Stimulation of Growth Hormone Secretion by Levodopa-Peopranolol in Children and Adolescents. Pediatrics 1975; 56: 262-266.

(18.) Giuffrida FMA, Berger K, Monte L, et al. Relationship between GH response and glycemic fluctuations in the glucagon stimulation test. Growth Hormone and IGF Research 2009; 19: 77-81.

(19.) Chanoine JP, Rebuffat E, Kahn A, Bergmann P, Van Vliet G. Glucose, Growth Hormone, Cortisol, and Insulin Responses to Glucagon Injection in Normal Infants, Aged 0.5-12 Months. Journal of Clinical Endocrinology and Metabolism 1995; 80: 3032-3035.

(20.) Biller BM, Samuels MH, Zagar A, et al. Sensitivity and Specificity of Six Tests for the Diagnosis of Adult GH Deficiency. Journal of Clinical Endocrinology and Metabolism 2002; 87: 2067-2079.

(21.) Conceicao FL, da Costa e Silva A, Leal Costa AJ, Vaisman M. Glucagon stimulation test for the diagnosis of GH deficiency in adults. Journal of Endocrinological investigation 2003; 26: 1065-1070.

(22.) Gomez JM, Espadero RM, Escobar-Jimenez F, et al. Growth hormone release after glucagon as a reliable test of growth hormone assessment in adults. Clinical Endocrinology (Oxf) 2002; 56: 329-334.

Mehmet Ali Eren, Suzan Tabur, Mehmet Nuri Turan *, Serpil Sarifakiogullari *, Tevfik Sabuncu

Harran University, Endocrinology and Metabolism, Sanliurfa, Turkey

* Harran University, Internal Medicine, Sanliurfa, Turkey

Address for Correspondence: Mehmet Ali Eren, MD, Harran University, Endocrinology and Metabolism, Sanliurfa, Turkey E-mail: drmalieren@hotmail.com Received: 22.12.2009 Accepted: 21.04.2010
Table 1. Clinical characteristics of the patients

Parameters Values

Chronological age * 14.9 [+ or -] 2.3
Bone age * 12.4 [+ or -] 2.8
n (male/female) 43 (32/11)
Height (cm) (+) 139.0 (111-158)
Weight (kg) (+) 34.0 (23.0-58.5)
BMI (kg/m2) (+) 16.6 (13.3-32.5)

BMI: body mass index; * values are means [+ or -] SD,
(+) values are medians (range)

Table 2. Response rates of the tests in the patients with
short stature

Tests Subject Response Percentage (%)

ITT 43 15 34.6
L-dopa test 32 11 34.4
Clonidine test 21 12 57.1
Glucagon test 12 2 16.7

ITT: insulin tolerance test

Table 3. Comparison response rates of L-dopa, clonidine and
glucagon tests with ITT

 L-dopa Clonidine

Test name Response No response Response No response

Response to ITT 4 (12.5) 7 (21.9) 4 (19) 1 (4.8)
No response to ITT 7 (21.9) 14 (43.8) 8 (38.1) 8 (38.1)
Total 11 (34.4) 21 (65.6) 12 (57.1) 9 (42.9)

 Glucagn

Test name Response No response

Response to ITT 1 (8.3) 2 (16.7)
No response to ITT 1 (8.3) 8 (66.7)
Total 2 (16.7) 10 (83.3)

ITT: insulin tolerance test; the results were given as n (%)

Table 4. Responses of the tests in the subjects with and without GHD

 ITT L-dopa

 Responsive No responsive Responsive No responsive

GHD (+) 2 21 1 16
GHD (-) 13 7 10 5

 Clonidine Glucagon

 Responsive No responsive Responsive No responsive

GHD (+) 3 8 0 10
GHD (-) 9 1 2 0

ITT: insulin tolerance test; GHD: growth hormone deficiency

Table 5. The sensitivity, specificity and predictivity rates of
the tests

 Positive Negative
 Sensitivity Specificity predictivity predictivity
Tests (%) (%) (%) (%)

ITT 91 65 75 86
Clonidine 72 90 85 75
L-dopa 94 66 76 90
Glucagon 100 100 100 100

ITT: insulin tolerance test
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Title Annotation:Original Article/Orijinal Makale
Author:Eren, Mehmet Ali; Tabur, Suzan; Turan, Mehmet Nuri; Sarifakiogullari, Serpil; Sabuncu, Tevfik
Publication:Turkish Journal of Endocrinology and Metabolism
Article Type:Report
Geographic Code:7TURK
Date:Mar 1, 2010
Words:2865
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