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Anterior and Posterior Pituitary Function Testing with Simultaneous Insulin Tolerance Test and a Novel Copeptin Assay

Departments of Endocrinology, Diabetology and Clinical Nutrition (M.C.-C., M.K., J.R., B.M.), and Neurology (M.K.), University Hospital Basel, CH-4031 Basel, Switzerland; Research Department (N.G.M.), B.R.A.H.M.S. AG, Biotechnology Centre, Hennigsdorf-Berlin, D-16761 Hennigsdorf, Germany; and Department of Endocrinology (K.C.S.D., G.E.B.), Christie Hospital, Manchester M20 4BX, United Kingdom

Address all correspondence and requests for reprints to: Mirjam Christ-Crain, Department of Endocrinology, University Hospitals, Petersgraben 4, CH-4031 Basel, Switzerland. E-mail: christmj{at}bluewin.ch.

	Abstract

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Context: Posterior pituitary function in patients with suspected diabetes insipidus is usually assessed by a water deprivation test. Alternatively, a nonosmotic stimulus such as hypoglycemia may be used to stimulate vasopressin [arginine vasopressin (AVP)] secretion. Plasma AVP measurement may aid in the diagnosis and, especially, differential diagnosis of diabetes insipidus and polydipsia. However, AVP measurement is cumbersome. Copeptin, the stable C-terminal glycopeptide of the AVP prohormone, is stoichiometrically secreted from the posterior pituitary.

Objective: The aim was to study the value of copeptin levels in the diagnosis of diabetes insipidus during insulin-induced hypoglycemia.

Patients and Methods: A total of 38 patients were studied during insulin-induced hypoglycemia as part of a combined pituitary function test for possible anterior pituitary disease. There were 29 patients who had normal posterior pituitary function, and nine had central diabetes insipidus. Blood sampling was done before and 30, 45, and 90 min after iv insulin injection. Copeptin was measured with a new sandwich immunoassay.

Results: Patients with intact posterior pituitary function had basal copeptin levels of 3.7 ± 1.5 pM, with a maximal increase to 11.1 ± 4.6 pM 45 min after insulin injection. Copeptin levels in patients with diabetes insipidus were 2.4 ± 0.5 pM before insulin injection, with a maximum increase to 3.7 ± 0.7 pM. Both basal and stimulated copeptin levels were lower in patients with diabetes insipidus as compared with patients with intact posterior pituitary function. A stimulated copeptin level 45 min after insulin injection of less than 4.75 pM had an optimal diagnostic accuracy to detect diabetes insipidus.

Conclusion: Copeptin measurement may be used to assess posterior together with anterior pituitary function during insulin-induced hypoglycemia.

	Introduction

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THE ANTIDIURETIC HORMONE arginine vasopressin (AVP) (also termed antidiuretic hormone) is synthesized in the hypothalamus and secreted by the neurohypophysis into the blood (1). Osmolality is the main physiological stimulus for AVP release. Insulin-induced hypoglycemia also stimulates AVP release (2, 3). Thus, insulin-induced hypoglycemia has been proposed as a helpful simultaneous test of anterior and posterior pituitary function by measuring AVP levels (4). Unfortunately, AVP measurement by RIA is cumbersome. More than 90% of AVP in the circulation is bound to platelets (5), resulting in either underestimation or in case of prolonged storage of unprocessed blood samples, in falsely elevated or varying AVP levels (5, 6). Furthermore, AVP is unstable in isolated plasma, even when stored at –20 C (7), and due to its small size, AVP cannot be measured by sandwich immunoassays but needs competitive assays, which are generally time consuming. Therefore, AVP measurement has not been implemented in clinical routine.

AVP derives from a precursor protein, pre-pro-vasopressin, which consists of a signal peptide, AVP, neurophysin II, and copeptin (8). Copeptin, the C-terminal part of the AVP precursor, is a 39-amino acid long glycosylated peptide with a leucine-rich core segment (9, 10). It is specific to the AVP-neurophysin II precursor but absent in the oxytocin-neurophysin I homolog. In neurosecretory granules, the AVP precursor is proteolytically processed to yield AVP, neurophysin II, and copeptin. The three peptides are stoichiometrically secreted from the posterior pituitary. Copeptin remains stable ex vivo for several days at room temperature in serum or plasma (11). A novel assay to measure copeptin and, thus, to quantify AVP secretion has recently been developed (11, 12). Here, we investigated the use of the copeptin assay in the diagnostic workup of patients with suspected diabetes insipidus.

	Patients and Methods

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Setting and study population

A total of 38 patients were studied during insulin-induced hypoglycemia as part of a combined pituitary function test for presumed anterior pituitary disease. There were 33 patients tested after transsphenoidal surgery (20 with pituitary adenomas, four with craniopharyngiomas, five with Rathke cleft cysts, three with other brain tumors, and one with hypophysitis) and five without transsphenoidal surgery (one with empty sella, one with hypoplasia of the pituitary, and three for other reasons). Of these, 29 patients had normal posterior pituitary function, and nine had well-established central diabetes insipidus, based on a water deprivation test that was performed as follows. The test started at 0800 h. From 0800–1800 h, fluid intake was not allowed. Blood sampling for plasma osmolality, sodium levels, urine osmolality, and measurement of the specific weight of urine, as well as measurement of blood pressure, heart rate, and weight of patients was done regularly. At 1600 h after an additional blood sampling, patients received desmopressin 2 µg iv and were allowed to drink at maximum 0.5 liter. At 1800 h (end of the test), patients were again tested for plasma and urine measurements and weight. The test was considered to be normal if after 8 h of thirsting, the ratio between urine and plasma osmolality increased more than 2.5 and weight decreased less than 2%, with a continuous decrease of urine volume and increase of specific weight of urine. A central diabetes insipidus was considered to be present when urine osmolality increased more than 50% after desmopressin was given.

The insulin-hypoglycemia test was performed as follows: blood sampling was done after an overnight fast. Patients were allowed to drink fluids apart from alcohol, tea, or coffee. An iv cannula was inserted 30 min before starting and was flushed with 10 ml saline after each sample had been obtained. Blood was drawn for basal measurement of blood glucose, cortisol, and copeptin levels. Insulin (0.1–0.2 U/kg) was injected iv. Additional blood was drawn 30, 45, and 90 min after insulin injection for repeat measurements of blood glucose, cortisol, and copeptin levels.

The study was approved by the local ethics committee for human studies, and written informed consent was obtained.

Copeptin was measured with a new sandwich immunoassay, as described (11, 12). This assay has an analytical detection limit of 1.7 pM. All samples were assayed as a batch analyses in one run.

Statistical analysis

Discrete variables are expressed as counts (percentage) and continuous variables as means ± SD unless stated otherwise. A two-group comparison of normally distributed data was performed by the Student’s t test. For data not normally distributed, the Mann-Whitney U test was used. Correlation analyses were performed using Spearman rank correlation. All testing was two-tailed, and P values < 0.05 were considered to indicate statistical significance.

	Results

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Baseline characteristics

A total of 29 patients had normal posterior pituitary function, and nine had central diabetes insipidus. Baseline characteristics, including anterior pituitary function, are shown in Table 1.

During the insulin hypoglycemia test, blood glucose decreased less than 2 mmol/liter in each patient, and the mean concentration decreased to (mean ± SD) 1.7 ± 0.8 mmol/liter. The maximum decrease in blood glucose was 30 min after insulin injection. Forty-five minutes after insulin injection, glucose levels were 2.0 ± 0.5 mmol/liter.

Patients with intact posterior pituitary function had basal copeptin levels of 3.7 ± 1.5 pM, with a significant increase 30 min after insulin injection to 7.3 ± 3.2 pM, 45 min after insulin injection to 11.1 ± 4.6, and 90 min after insulin injection to 7.1 ± 4.5 pM (P < 0.001) (Fig. 1).

View larger version (19K): [in this window] [in a new window]   FIG. 1. Copeptin levels basally, and 30, 45, and 90 min after insulin injection in the nine patients with diabetes insipidus (left side) and in the 29 patients with intact posterior pituitary dysfunction (right side). In the upper panel, all data are plotted. In the lower panel, data are shown as median and SDs.

Basal and stimulated copeptin levels were significantly lower in patients with diabetes insipidus compared with patients with intact posterior pituitary function (P = 0.003 and <0.001, respectively).

A basal copeptin level less than 2.59 pM had an 89% sensitivity and 76% specificity to detect diabetes insipidus. A stimulated copeptin level 45 min after insulin injection less than 4.75 pM had a 100% sensitivity and 100% specificity to detect diabetes insipidus (Fig. 2). Copeptin levels 30 and 90 min after insulin injection had an area under the receiver operating curve to detect diabetes insipidus of 0.94 and 0.98, respectively.

View larger version (11K): [in this window] [in a new window]   FIG. 2. Receiver operating curve analysis is shown of basal and stimulated copeptin levels 45 min after insulin injection to predict diabetes insipidus. Sensitivity is calculated with patients with diabetes insipidus (n = 9) and specificity with patients without diabetes insipidus (n = 29). AUC, Area under the curve.

	Discussion

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Copeptin, which is co-secreted in an equimolar ratio to AVP, has been studied in healthy volunteers and critically ill patients (11, 12). Thereby, copeptin values correlated strongly with AVP levels (r = 0.83) (11). Thus, it can be used as a measure for the actual release of AVP gene products.

In our postoperative cohort of patients, copeptin levels discriminate between patients with and without diabetes insipidus. Thereby, basal copeptin levels had a good, albeit not perfect, discriminatory capacity. In contrast, stimulated copeptin levels measured 45 min after insulin injection had the best sensitivity and specificity to detect diabetes insipidus. This is in accordance with the first report about plasma AVP levels during insulin-induced hypoglycemia to test posterior pituitary function.

Insulin-induced hypoglycemia is a standardized procedure to assess the individual stress response, namely the integrity of the hypothalamic-pituitary-adrenal axis. Thereby, as a broad and unspecific stressor, hypoglycemia induces the release of several pituitary hormones. AVP is known to be released not only in response to osmotic stimuli, but also after a hypoglycemic stimulus (2, 3). In the rat, hypoglycemia induces AVP release from both parvocellular and magnocellular neurons (14, 15). However, the precise location where AVP in humans is released due to osmotic and nonosmotic stimuli, as well as the interaction between magnocellular and parvocellular neurons, remains to be clarified. In our small study cohort, we had no false-positive or false-negative copeptin values arguing for a release from both structures. However, this has to be confirmed in a larger cohort of patients.

Our report has limitations. First, a challenge is the detection of partial diabetes insipidus (16), which was not investigated in this study. Second, AVP was not measured to show directly the correlation between copeptin and AVP levels. Also, for the diagnosis of diabetes insipidus, AVP measurements are formally required. However, copeptin is secreted stoichiometrically with AVP (11). Furthermore, AVP levels are needed primarily to distinguish between complete and partial forms of diabetes insipidus. Our patients had complete diabetes insipidus based on their inability to concentrate their urine more than 2.5 times the plasma osmolarity, which is commonly accepted as a diagnostic criterion with a false-positive rate of only 10% (17).

The measurement of neurophysin II levels would hold the same potential benefit compared with copeptin, yet, has not reached the point of clinical use. In particular, there is a considerable sequence homology between neurophysin II and the oxytocin-associated neurophysin I, which may compromise assay specificity. Conversely, copeptin is lacking the oxytocin precursor, thus constituting a readily measurable serum parameter that specifically reflects vasopressin secretion.

In conclusion, the measurement of copeptin concentrations during insulin-induced hypoglycemia may be a useful clinical test of posterior pituitary function. If confirmed in a larger patient cohort, including partial diabetes insipidus, it may be used in patients after pituitary surgery to test anterior and posterior pituitary function simultaneously, and thereby to facilitate the postoperative assessment of patients after transsphenoidal surgery.

	Acknowledgments

 
We thank Anne Schmiedel, Frank Bonconseil, Uwe Zingler, and Barbara Schäffus (B.R.A.H.M.S. AG, Hennigsdorf, Germany) for excellent technical assistance with the copeptin assay. We also thank the staff of the Endocrinology Department, University Hospital Basel, for its most helpful support during the study.

	Footnotes

 
Total study costs were granted by funds from the Department of Endocrinology and a grant from the Swiss Foundation of Medical and Biological Stipends (SSMBS) (PASMA-114617/1, to M.C.-C.).

Disclosure Statement: M.K., N.G.M., K.C.S.D., J.R., G.E.B., B.M., and M.C.-C. have nothing to declare.

First Published Online April 10, 2007

Abbreviation: AVP, Arginine vasopressin.

Received September 20, 2006.

Accepted April 4, 2007.

	References

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This Article Abstract Full Text (PDF) All Versions of this Article: 92/7/2640    most recent Author Manuscript (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Katan, M. Articles by Christ-Crain, M. Search for Related Content PubMed PubMed Citation Articles by Katan, M. Articles by Christ-Crain, M. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Medline Plus Health Information Pituitary Disorders Related Collections Neuroendocrinology and Pituitary