Combining Growth Hormone Releasing Hormone- Arginine and ...

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The Journal of Clinical Endocrinology & Metabolism 92(3):853– 856 Copyright © 2007 by The Endocrine Society doi: 10.1210/jc.2006-2140

Combining Growth Hormone Releasing HormoneArginine and Synacthen Testing Diminishes the Cortisol Response S. Siyambalapitiya, V. Ibbotson, A. Doane, E. Ghigo, M. J. Campbell, and R. J. Ross Endocrinology and Reproduction Section (S.S., V.I., A.D., R.J.R.), and Medical Statistics Group (M.J.C.), The School of Health and Related Research, University of Sheffield, Sheffield S10 2JF, United Kingdom; and University of Turin (E.G.), 10060 Turin, Italy Objectives: The GHRH/arginine test and short synacthen test (SST) have been validated as safe alternatives to the insulin tolerance test for the assessment of the GH reserve and hypothalamic-pituitaryadrenal axis integrity, respectively. However, these two tests are usually performed separately. The objective was to see whether the synacthen and GHRH/arginine tests could be combined to save time and blood samples and minimize inconvenience to patients.

GHRH/arginine was given with synacthen, patients had a lower peak cortisol response with a mean difference of 116 nmol/liter (95% confidence interval, 52.54 to 179.37; P ⬍ 0.001), and one patient with a normal response on the SST had a subnormal cortisol response in the combined test. Similar lower peak cortisol responses were observed in males and females with combined test. The difference between the peak cortisol responses showed no significant correlation with age (r ⫽ 0.123; P ⫽ 0.58) or with the body mass index (r ⫽ ⫺0.376; P ⫽ 0.09). There was no difference in GH measurements between the GHRH/arginine test done alone or in combination with the SST.

Patients/Methods: Twenty-four consecutive patients with adult onset pituitary disease requiring pituitary function testing were randomized to receive sequentially and in random order a SST, a GHRH/ arginine test, and a combined SST and GHRH/arginine test on three different visits separated by at least 1 wk.

Conclusions: Combining the SST and GHRH/arginine test results in a lower cortisol response to synacthen. For this reason, the combined test cannot be recommended to assess the integrity of cortisol and GH reserve using current diagnostic criteria. (J Clin Endocrinol Metab 92: 853– 856, 2007)

Results: There was no difference in basal cortisol or ACTH values for the SST done alone or during the combined test. However, when

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N PATIENTS WITH pituitary disease, the diagnosis of GH and hypothalamic-pituitary-adrenal (HPA) axis deficiency is made using dynamic pituitary function tests (1, 2). The Growth Hormone Research Society recommends the insulin tolerance test (ITT) as the test of choice for the assessment of GH reserve (2). An advantage of the ITT is its ability to assess the adequacy of GH and ACTH reserve simultaneously. The ITT is safe when carried out by experienced staff (3). However, it is unpleasant for the patient, is not suitable in elderly patients, and carries a risk of seizure or myocardial ischemia in patients with a history of epilepsy or severe coronary artery disease. The glucagon stimulation test is an alternative to the ITT; however, it is a less powerful stimulus and has relatively poor specificity (4). Thus, many units use a variety of individual tests for GH and cortisol performed on separate days, which is time consuming and inconvenient. We have examined whether a test of GH secretion can be combined with a test of cortisol reserve without interaction. The sequential administration of GHRH with l-arginine (GHRH/arginine test) is a potent stimulus of GH secretion

and has become the most commonly used test in the United States (5). The GH response is reproducible and independent of age and shows limited inter- and intraindividual variability (6). The test can be performed in 60 min with only two blood samples. The short synacthen test (SST) has been validated and widely used to assess the integrity of HPA axis in patients with pituitary disease (7–12). It has been shown to be equivalent to the ITT in assessing secondary hypoadrenalism with a 97% positive predictive value in excluding ACTH deficiency (7), and it has become the investigation of choice in many endocrine centers. The objective of this study was to see whether the SST and GHRH/arginine tests could be combined to save time and blood samples and to minimize inconvenience to the patient. Subjects and Methods Subjects The study was approved by the North Sheffield Ethics Committee, and written informed consent was obtained from all patients. The study group consisted of 24 consecutive patients with adult onset hypothalamic-pituitary disease who required anterior pituitary function testing. A sample size of 24 was selected using the three-over-n rule, whereby a difference of more than 12% (3 of 24) is deemed clinically significant at the 95% confidence level (CI) (13). Patient characteristics are given in Table 1. Patients with asthma or epilepsy and those on GH, hydrocortisone, or estrogen therapy were excluded from the study.

First Published Online December 27, 2006 Abbreviations: BMI, Body mass index; CI, confidence interval; HPA, hypothalamic-pituitary-adrenal; ITT, insulin tolerance test; SST, short synacthen test. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

Study procedure Patients were randomized according to a Latin square design to receive sequentially and in random order a SST, a GHRH/arginine test

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J Clin Endocrinol Metab, March 2007, 92(3):853– 856

Siyambalapitiya et al. • Combined Pituitary Test

TABLE 1. Patient characteristics Pituitary hormone deficiencies

Patient

Gender

Age (yr)

BMI (kg/m2)

Diagnosis/reason for investigation

IGF-I (␮g/liter)

GH

FSH/LH

TSH

ACTH

Vasopressin

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

F M M F M M M M F M F M F F M M M M M F M M F F

41 42 45 39 47 60 64 51 54 75 56 58 31 65 43 51 53 67 65 39 62 69 76 35

38.1 20.4 31.5 21.5 15.7 32.5 28.1 26.8 45.5 26.8 45.1 26.9 29.1 30 32.3 23.5 39.1 24.8 24.9 27.4 24.6 45.2 28.7 38.7

Low IGF-1 Isolated GH deficiency NFPA after surgery and DXT Idiopathic Low IGF-1 NFPA NFPA NFPA after surgery and DXT Isolated GH deficiency NFPA NFPA after surgery and DXT Idiopathic Medulloblastoma after surgery and DXT NFPA NFPA NFPA after surgery NFPA NFPA NFPA after surgery and DXT Pituitary cyst Macro-prolactinoma Idiopathic NFPA DXT for ALL

48 (95–343) 36 (92–284) 204 (80 –276) 84 (95–343) 78 (80 –276) 155 (60 –224) 202 (36 –226) 54 (78 –248) 48 (37–231) 60 (40 –204) 28 (65–251) 209 (60 –224) 43 (102– 491) 65 (52–227) 107 (80 –276) 100 (78 –248 126 (61–285) 193 (52–227) 91 (52–227) 139 (95–343) 63 (50 –255) 91 (52–227) 78 (40 –204) 159 (96 –302)

N Y N Y N Y Y Y Y Y Y Y Y Y Y Y Y Y Y N Y Y Y Y

N N Y Y N Y N Y N Y N N Y N Y Y Y Y Y N Y Y N N

N N N N N Y N N N Y N N Y N Y N N N Y Y Y N N N

N N N N N N N N N N N N N N N N N N N N N N N N

N N N N N N N N N N N N N N N N N Y N N N N N N

NFPA, Non-functioning pituitary adenoma; DXT, radiotherapy; ALL, acute lymphoblastic leukemia; F, female; M, male; Y, yes; N, no. In the IGF-I column, age-related reference ranges are in parentheses. and a combined SST and GHRH/arginine test. Patients underwent the three different tests on three different visits separated by at least 1 wk. The tests were performed as follows: 1) SST: 250 ␮g im synacthen was given at time zero. Blood samples for cortisol were withdrawn before and 30 min after synacthen administration. ACTH was measured basally. 2) GHRH/arginine test: GHRH (GEREF diagnostic, provided by Serono, Middlesex, UK) 1 mg/kg was given as an iv bolus at time zero, followed immediately by a 30-min infusion of l-arginine (30 g) in 100 ml normal saline. Blood samples were taken at 0, 30, 45, and 60 min for serum GH. 3) Combined (SST and GHRH/arginine) test: This was performed as above for individual tests, but all drugs were administered at time zero, and the same sampling regimens were used. Serum cortisol was analyzed using the Bayer Advia Centaur Automated Immunoassay System (Bayer Diagnostics, Tarrytown, NY). The interassay coefficient of variation was 7% at 200 nmol/liter and 8% at 1050 nmol/liter. Plasma ACTH was analyzed using the DPC Immulite 2000 (Diagnostic Products Corp., Los Angeles, CA) with an interassay coefficient of variation of 4% at 28 ng/liter. GH was analyzed using Immulite GH (Diagnostic Products Corp.). Intraassay and interassay coefficients of variation for GH assay were 5.3– 6.5 and 5.5– 6.1%, respectively.

Statistical analysis A peak serum GH value of more than 27 mU/liter (9 ng/ml) and a peak cortisol value of more than 550 nmol/liter (19.9 ␮g/dl) were considered a normal response to the GH and cortisol stimulation tests. The peak responses to the three stimulation tests were used as the primary variables for analysis. Statistical comparisons of mean basal (time zero), mean peak cortisol levels (time 30 min), and mean peak GH

levels (time 30, 45, or 60 min) during the single (SST alone or GHRH/ arginine alone) and combined test were performed using the t test for paired data. A level of statistical significance was set at P ⬍ 0.05. Linear regression analysis was used to assess the relationship of age and body mass index (BMI) (independent variables) with mean difference in peak cortisol and GH levels between the single and combined tests (dependent variable).

Results

From a practical perspective, there were no problems combining the two tests. There were no serious side effects observed during the study, except on one occasion where a patient developed a vasovagal attack. The relevant patient was withdrawn from the study and replaced with another patient. Cortisol responses (Table 2 and Fig. 1)

There was no difference in basal cortisol or ACTH values for the SST done alone or during the combined test. However, when GHRH/arginine was given with synacthen, patients had a significantly lower peak cortisol response with a mean difference of 116 nmol/liter (95% CI, 52.54 to 179.37; P ⬍ 0.001). All 24 patients had a peak cortisol response above the cutoff level of 550 nmol/liter with the SST alone. One patient failed to achieve the peak cortisol response above the cutoff level of 550 nmol/liter during the combined test.

TABLE 2. Mean (SD) ACTH and cortisol and mean difference (95% CI) in ACTH and cortisol response to SST alone and combined test

SST alone Combined SST and GHRH/arginine test ACTH and cortisol differences a

P ⬍ 0.001 SST alone vs. combined.

Mean basal ACTH (pg/ml)

Mean basal cortisol (nmol/liter)

26.6 (14.08) 30.4 (19.67) ⫺3.8 (⫺10.57 to 2.92)

419 (157.34) 403.3 (158.89) 15.7 (24.59 to 56)

Mean peak cortisol (nmol/liter)

846.6 (222.90) 730.6 (111.32) 116 (52.54 to 179.37)a



FIG. 1. The box plot represents the peak cortisol response and cortisol difference to SST alone and combined test. The dotted line represents the cortisol cutoff for the SST. The symbols represent the outliers and the number next to them the patient identifier according to Table 1.

Similar lower peak cortisol responses were observed in males and females with the combined test compared with the synacthen test alone. The difference between the mean peak cortisol responses of SST alone and combined test showed no significant correlation with age (r ⫽ 0.123; P ⫽ 0.58) or BMI (r ⫽ ⫺0.376; P ⫽ 0.09). GH responses (Table 3 and Fig. 2)

There was no significant difference between the GH measurements at any time point or the peak value between the GHRH/arginine test alone or when combined. Peak mean GH difference was ⫺2.6 mU/liter (95% CI, ⫺6.24 to 0.99; P ⫽ 0.15). Only three of 24 patients had a peak GH response above the cutoff level of 27 mU/liter with GHRH/arginine test alone. There were four patients who had a peak GH response above the cutoff with the combined test showing almost a similar clinical outcome. Discussion

The GHRH/arginine and SST are commonly used for the assessment of GH and cortisol reserve, respectively. Currently, patients attend on 2 separate days for the tests and have to undergo cannulation on two separate occasions with associated inconvenience and cost. We have investigated whether the two tests could be combined. There were no practical difficulties combining the two tests. There were no differences in GH levels whether the GHRH/arginine test was done alone or combined with synacthen. However, cortisol levels were significantly lower during the combined test compared with the SST alone. This would have led to one

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FIG. 2. The box plot represents the peak GH response and GH difference to GHRH/arginine test alone and combined test. The dotted line represents the GH cutoff level for the GHRH/arginine test. The symbols represent the outliers and the number next to them the patient identifier according to Table 1.

patient being classified as cortisol deficient who had a normal response to SST alone. There are limited data on the interaction between ACTH and GH release. In healthy volunteers, there is a significant rise in GH 30 to 45 min after iv administration of synthetic ACTH (14). In another study, the response of GH to synacthen was only seen in healthy females, but males exhibited a similar rise when they were pretreated with estrogens (15). We found no effect of synacthen on the GH response in our patients. This could possibly relate to the fact that the majority of our patients were GH deficient based on their responses. In our cohort, 33% had isolated GH deficiency. We accept that this may not reflect the normal incidence of isolated GH deficiency in adult patients because in this study, we deliberately chose patients who had normal cortisol reserve until the time of testing and required testing of their GH axis. Our patients had a high average BMI, and this could have influenced the GH response. High BMI is known to reduce the GH response to GH stimulation tests (16, 17). The interaction between GH secretagogues and ACTH release has been studied in detail. Both ghrelin and the synthetic GH secretagogue hexarelin stimulate ACTH and hence cortisol release (18, 19). This is thought to be predominantly via arginine vasopressin release, with a lesser effect on CRH (20). In contrast, GHRH is not known to affect ACTH or adrenal cortisol release (19), and we expected no interaction of GHRH with the cortisol response to SST. l-Arginine is an essential amino acid that is used in provocative testing of GH secretion. There is evidence that l-arginine acts through inhibition of somatostatin release and also through stimulation of GHRH secretion (21, 22). Previous studies have not shown

TABLE 3. Mean (SD) GH and mean difference (95% CI) in GH response to GHRH and arginine test alone and combined test

GHRH/arginine test Combined test GH response difference

0 min GH (mU/liter)

30 min GH (mU/liter)



Peak GH (mU/liter)

1.5 (2.26) 1.1 (1.13) 0.4 (⫺0.34 to 1.04)

11.5 (17.14) 14.2 (16.53) ⫺2.7 (⫺7.12 to 1.73)

15.3 (21.62) 16.9 (16.89) ⫺1.7 (⫺6.89 to 3.55)

14.5 (17.86) 16.9 (22.60) ⫺2.4 (⫺5.87 to 1.08)

16.8 (21.54) 19.4 (22.65) ⫺2.6 (⫺6.24 to 0.99)

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any significant influence of arginine on pituitary ACTH or adrenal cortisol response during GH assessment using larginine (23). Thus, previous evidence suggested that neither GHRH nor l-arginine would influence the cortisol response to synacthen. However, we observed that cortisol response to synacthen was significantly lower when combined with GHRH/arginine, and in one patient, this would have resulted in misclassifying the patient as cortisol deficient. One possibility is that the GHRH/arginine could have delayed the cortisol peak for synacthen stimulation; however, our study did not address this point. In conclusion, combining the SST and GHRH/arginine test results in a lower cortisol response to synacthen and for this reason the combined test cannot be recommended to assess the integrity of cortisol and GH reserve using current criteria for a normal response. Further studies are necessary to determine the mechanism of interaction between synacthen and GHRH/arginine stimulation and also to investigate the possibility of using a combined test to assess the cortisol reserve with a lower cortisol cutoff level. Acknowledgments We are grateful to Richard Barker and Serono for technical support and an educational grant that supported this work. Received September 29, 2006. Accepted December 15, 2006. Address all correspondence and requests for reprints to: Professor Richard J. M. Ross, University of Sheffield, Room 112, Floor M, Royal Hallamshire Hospital, Glossop Road, Sheffield S10 2JF, United Kingdom. E-mail: [email protected]. Disclosure Statement: The authors have nothing to disclose.

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JCEM is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.