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Copeptin as a biomarker and a diagnostic tool in the evaluation of patients with polyuria–polydipsia and hyponatremia
Accepted Manuscript Copeptin as a biomarker and a diagnostic tool in the evaluation of patients with polyuria-polydipsia and hyponatremia M. Christ-Crain, MD, PhD, Prof., N.G. Morgenthaler, MD, PhD, P.D.W. Fenske, MD, PhD PII:
S1521-690X(16)00008-7
DOI:
10.1016/j.beem.2016.02.003
Reference:
YBEEM 1077
To appear in:
Best Practice & Research Clinical Endocrinology & Metabolism
Please cite this article as: Christ-Crain M, Morgenthaler NG, Fenske PW, Copeptin as a biomarker and a diagnostic tool in the evaluation of patients with polyuria-polydipsia and hyponatremia, Best Practice & Research Clinical Endocrinology & Metabolism (2016), doi: 10.1016/j.beem.2016.02.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title Page
Copeptin as a biomarker and a diagnostic tool in the evaluation of patients with polyuria-polydipsia and hyponatremia
Prof. M. Christ-Crain, MD, PhD (corresponding author)
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Authors:
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Department of Endocrinology, University hospital Basel, University of Basel, Switzerland Email: [email protected]; Tel. +41 61 328 7080 Fax. +41 61 265 5100
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N. G. Morgenthaler, MD, PhD Institut für Experimentelle Endokrinologie, Charité-Universitätsmedizin-Berlin , Berlin, Germany Email: [email protected] Tel. +49 42122051610
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PD W. Fenske, MD, PhD
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Leipzig University Medical Center, Integrated Research and Treatment Center for Adiposity Diseases Email: [email protected] Tel: +49 341 9713306
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ACCEPTED MANUSCRIPT Abstract Copeptin is part of the 164 amino acid precursor protein preprovasopressin together with vasopressin and neurophysin II. During precursor processing, copeptin is released together with vasopressin. Copeptin concentrations respond as rapidly as vasopressin to changes in
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osmolality, a decrease in blood pressure or stress and there is a close correlation of vasopressin and copeptin concentrations. For these reasons, copeptin is propagated as a surrogate marker for vasopressin in the differential diagnosis of the polyuria-polydipsia
syndromes and hyponatremia. Results of prospective studies show that a baseline copeptin level without prior fluid deprivation >20 pmol/L is able to identify patients with nephrogenic
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diabetes insipidus, whereas osmotically stimulated copeptin levels differentiate between patients with partial central diabetes insipidus and primary polydipsia with a high sensitivity
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and specificity >94%. In hyponatremia, low copeptin levels point to primary polydipsia and high levels to hypovolemic hyponatremia. The copeptin to urinary sodium ratio differentiates accurately between volume-depleted and normovolemic disorders.
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Key words: Arginine Vasopressin, copeptin, polyuria-polydipsia syndrome, diabetes insipidus, hyponatremia
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ACCEPTED MANUSCRIPT Background: Copeptin Synthesis and function of copeptin Copeptin is part of the 164 amino acid precursor protein preprovasopressin together with vasopressin (AVP) and neurophysin II. It is a glycosylated peptide of 39 amino acids with a leucine-rich core region (1, 2). During precursor processing, copeptin is released together
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with AVP from magno- and parvocellular neurons of the hypothalamus. The molecular mass of copeptin is around 5 kDa which is in accordance with the theoretical prediction of processed copeptin (3) (Figure 1).
In 1972, Holwerda first described a 39 amino acid glycopeptide from the posterior pituitary of
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pigs (4). Levy et al. later termed this glycopeptide “copeptin” (2). It remained relatively unknown except to a few experts in the field of diagnostics until it was characterized in 2006 as a potential biomarker (3). Since the development of the first copeptin assay, the number
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of original publications on copeptin increases every year almost exponentially. In contrast to AVP, the physiological function of copeptin is still not fully understood. The other precursor fragment neurophysin II acts as a carrier protein in the transport of AVP from the hypothalamus to the neurohypophysis, and some experiments suggested the role of copeptin as a prolactin-releasing factor, albeit with inconclusive results (5, 6). Other work indicates that copeptin may play a role in the correct structural formation of proAVP (7).
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Reports on the interaction with the calnexin/ calreticulin system (8, 9), which monitors protein folding and interacts with glycosylated proteins, suggest a function as a chaperone, decreasing the formation of inactive, and increasing the formation of active hormones. It is tempting to attribute the inefficient monomer folding in the absence of copeptin to the
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pathogenesis of central diabetes insipidus, but this needs further examination. Also, it does not explain why oxytocin, the other neurohormone with similar structure to AVP, does not require a copeptin-like carboxy-terminal fragment for correct maturation.
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Copeptin response to osmotic and fluid status in healthy volunteers It is known that the copeptin concentration rises as rapidly as AVP to changes in osmolality, a decrease in blood pressure or to unspecific stress. This increase can be explained by the equimolar production together with AVP. The fact that copeptin is stable in serum or plasma for days, once the blood sample is removed from the body, indicates that copeptin is either processed by tissue-bound proteases, and/or rapidly eliminated via the kidneys or the liver. Copeptin does not accumulate in the body as an irrelevant side product of AVP synthesis, but no specific copeptin receptor or copeptin elimination mechanism has been identified. Evidence for a partial renal elimination is provided by copeptin measurements in the urine.
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ACCEPTED MANUSCRIPT A direct comparison between copeptin and AVP serum concentrations in relationship to serum osmolality was done in collaboration with a group of AVP experts (10) (Figure 2). In this study, plasma osmolality, copeptin, and AVP levels were measured in 20 volunteers at baseline, after an oral water load, and during and after i.v. infusion of 3% saline. During the experiment, median plasma osmolality decreased from 290 mOsm/kg (range, 284–302) at baseline to 281 (273–288) mOsm/kg after water load and rose to 301 (298–307) mOsm/kg
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after hypertonic saline. At the same time, median plasma copeptin concentrations decreased from 3.3 (1.1–36.4) pmol/L at baseline to 2.0 (0.9 –10.4) pmol/L after water load and increased to 13.6 (3.7– 43.3) pmol/L after hypertonic saline. The correlation between copeptin and serum osmolality was stronger (Spearman’s rank correlation coefficient r=0.77)
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than between AVP and serum osmolality (r=0.49). There was a close correlation between AVP and copeptin concentrations (r=0.8). The strength of the correlation between AVP and copeptin depends on the quality of the AVP assay used, but most data show a correlation as
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would be expected from the equimolar release of both peptides (3).
Technical Advantages of Copeptin Measurement compared to AVP Measurement of mature AVP is difficult due to its small molecular size and subject to preanalytical errors. The small size prevents the efficient binding of two antibodies at the same time, which would be needed for sensitive sandwich immunoassays. Preanalytical errors
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include the strong binding of AVP to thrombocytes in the serum (due to the presence of vasopressin-receptors), and the short half-life time in vivo (3, 11). In contrast, for copeptin two assays are available with sufficient technical description and clinical data justifying their routine clinical use. These are the original sandwich immunoluminometric assay (LIA) (3) and
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its automated immunofluorescent successor (on the KRYPTOR platform). For both assays, CE IVD (European Community In Vitro Diagnostics) approval is available, but FDA (United States Federal Drug Administration) approval is still pending.
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The advantages of copeptin measurement compared to AVP mainly involve (i) the lower minimally required volume of only 50 µL serum or plasma, (ii) no
requirements for an
extraction step or other pre-analytical procedures such as the addition of protease inhibitors, (iii) results are in general available in approximately 0.5 to 2.5 hours, and (iv) as a sandwich immunoassay, it is more sensitive than competitive AVP immunoassays; (v) finally, copeptin, unlike mature AVP, is much more stable in plasma or serum ex vivo. Ex vivo stability of copeptin (<20% loss of recovery) was shown for serum and plasma for at least seven days at room temperature and at 14 days at 4°C.
Normal values and physiological response of plasma copeptin in healthy volunteers
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ACCEPTED MANUSCRIPT The reference range for copeptin was first defined in a study with 359 healthy volunteers. This study showed the broad range of copeptin values. The median copeptin plasma concentration was 4.2 pmol/L with a range between 1- 13.8 pmol/L (3). Similarly, copeptin concentrations in healthy volunteers of a population of 5000 individuals ranged between 1 and 13 pmol/L (upper 97.5 percentile) with median values <5 pmol/L (12). Men consistently show slightly higher values than women, but the difference in median values is only about 1
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pmol/L (12). Copeptin plasma concentration shows no correlation with age (3). The circadian rhythm of copeptin has only been investigated in a small study in 7 healthy volunteers, showing no consistent circadian rhythm (13).
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It is important to note that copeptin may fluctuate according to individual physiological conditions. In a study of 24 healthy volunteers, water deprivation over 28 hours increased serum copeptin from 4.6±1.7 pmol/L to 9.2±5.2 pmol/L (p<0.001). Additional infusion of
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hypertonic saline further increased plasma copeptin from 4.9±3.0 pmol/L to 19.9±4.8 pmol/L (p<0.001). Conversely, copeptin decreased from 6.2±2.4 pmol/L to 2.4±2.1 pmol/L (p<0.01) during hypotonic saline infusion (14). Essentially, copeptin showed identical changes during disordered water states or osmolality as previously shown for AVP.
In response to exercise, the copeptin concentration in blood increases, but does not exceed
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the 99th percentile of the reference range (15, 16).
In contrast, copeptin levels seem not to be affected by food intake: in a study in 30 healthy volunteers undergoing an oral glucose tolerance test and a mixed meal tolerance test, median copeptin levels remained unchanged or even decreased slightly (from 4.9 [3.6–8.3]
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and 4.9 [3.6–7.1] pmol/l to 3.2 (2.8–5.9) and 4.1 (2.7–6.1) pmol/l after the respective tests). This suggests that copeptin levels can be interpreted independently of the prandial status (17). In contrast, already small amounts of fluid (200-300 ml) seem to significantly decrease
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copeptin levels, underlining that any recent fluid intake must be considered when interpreting copeptin levels (17).
Copeptin response to unspecific stressors As for AVP, the main stimulus for copeptin is an increase in osmolality or a decrease in plasma volume. In addition, somatic stress, e.g. due to disease, is a major determinant of copeptin levels. Copeptin was shown to more subtly mirror the individual stress level as compared to cortisol. Accordingly, copeptin increases with increasing severity of disease in pneumonia, ischemic stroke and other diseases (18-20). Due to this positive association of copeptin with the severity of illness, copeptin has been proposed as a prognostic marker in acute illness. As such, copeptin has been analyzed in sepsis, pneumonia, lower respiratory 5
ACCEPTED MANUSCRIPT tract infections, stroke and other acute illnesses and was found to discriminate patients with unfavorable outcomes from patients with favourable outcomes (18-20). Moreover, copeptin improves the prognostic information provided by commonly used clinical scoring instruments. An accurate prognostic assessment has the potential to guide interventions and effectively plan and monitor rehabilitation and, thus optimize the management of individual patients and
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the allocation of limited health care resources.
In contrast to stress associated with somatic disease little is known about the influence of psychological stress on copeptin levels. Two recent studies in healthy volunteers undergoing a trier social stress test (TSST), and in medical students before and after a written medical
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exam, respectively have shown that copeptin increases (to a median of around 5 pmol/L, baseline 3.7 pmol/L) upon psychological stress, to a much smaller degree than observed in response to somatic disease (21, 22). This points to the fact that psychological stress has to
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be taken into account as a confounding factor in disorders with only subtly abnormal copeptin levels, e.g. in polyuria-polydipsia syndromes. However, in acute conditions like pneumonia or acute myocardial infarction, where copeptin levels often exceed the upper limit of the normal distribution, the modest increase seen in psychological stress is of lesser relevance. In contrast to cortisol, copeptin did not seem to predict examination performance in medical students (22).
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Interestingly, patients with complete central diabetes insipidus showed no increase upon psychological stress (peak 2.15 pmol/L (1.5–2.28)) in contrast to healthy volunteers, where copeptin levels increased significantly to 5.1 (3.2–7.0) pmol/L. By contrast, cortisol values were similar in patients and volunteers. This shows that the copeptin response to stress is
Copeptin as a biomarker in polyuria-polydipsia syndromes
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blunted in patients with diabetes insipidus (20).
The fact that copeptin mirrors AVP concentrations in the circulation predestines it as a biomarker in AVP-dependent diseases, especially in polyuria-polydipsia syndromes and hyponatremia. In the following, this review article will focus on copeptin as a diagnostic biomarker in these two specific entities. A spectrum of diseases like central diabetes insipidus, nephrogenic diabetes insipidus and primary polydipsia clinically present with polyuria and polydipsia (23). Careful differentiation of the underlying disorders is mandatory as treatment strategies vary substantially. After ruling out other, AVP-independent etiologies, such as hyperglycemia and hypercalcemia, the precise differentiation is often difficult to achieve (24). The available tests are not satisfactory (25-28) and often result in false diagnosis, especially in patients with 6
ACCEPTED MANUSCRIPT primary polydipsia and mild forms of diabetes insipidus (29-32). The widely accepted “goldstandard” in the differential diagnosis of polyuria/polydipsia is the water deprivation test with indirect assessment of the AVP activity by measurement of the maximally reached urine osmolality during a prolonged dehydration period or with direct measurement of AVP. However, recent findings revealed dramatic limitations of this procedure with an overall diagnostic accuracy of 70%, and an accuracy of only 41% in patients with primary polydipsia
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(29). Several reasons may account for this limited diagnostic outcome. Firstly, an accurate definition of the area defining the normal physiological relationship between plasma AVP and serum osmolality as an important prerequisite for the test interpretation, has long been missed (33-35), and accordingly, the biological variation of osmotic regulation and the non-
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linear association between plasma AVP and serum osmolality have not been accurately defined (29). Secondly, as mentioned above, the AVP assay per se is subject to several technical limitations, resulting in a high pre-analytical instability (3, 36, 37).
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Therefore, the availability of a novel immunoassay to measure copeptin has led to investigations whether measurement of copeptin can improve the differential diagnosis in patients with polyuria/polydipsia.
A first study in 2007 showed that hypoglycemia-induced copeptin response is a highly useful measure to identify patients with complete diabetes insipidus following transphenoidal pituitary surgery (38). In this study, patients with intact posterior pituitary function had basal
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copeptin levels of 3.7 ± 1.5 pmol/L with a maximal increase to 11.1 ± 4.6 pmol/L at 45 minutes after insulin injection. Copeptin levels in patients with diabetes insipidus were 2.4 ± 0.5 pmol/L before insulin injection with a maximum increase to 3.7 ± 0.7 pmol/L. Both basal and stimulated copeptin levels were lower in patients with central diabetes insipidus as
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compared to patients with intact posterior pituitary function. A stimulated copeptin level 45 minutes after insulin injection of less than 4.75 pmol/L had an optimal diagnostic accuracy to detect diabetes insipidus.
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Most recently, in the same population of patients undergoing pituitary surgery, copeptin levels measured at the day of surgery were shown to predict later diabetes insipidus (39). This study included 205 patients undergoing pituitary surgery at three Swiss or Canadian referral centers. Copeptin was measured pre-operatively and on each postoperative day until discharge. Fifty (24.4%) patients developed postoperative diabetes insipidus. Post-surgically, median copeptin levels in patients developing diabetes insipidus did not increase, and were lower than in patients without diabetes insipidus: 2.9 pmol/L vs 10.8 pmol/L, p<0.001. Logistic regression analysis showed a strong association of postoperative copeptin concentrations and diabetes insipidus, even after considering known predisposing factors for this complication (adjusted odds ratio (95% confidence interval) 1.41 (1.16–1.73). In patients with postoperative copeptin values <2.5 pmol/L, the positive predictive value for diabetes 7
ACCEPTED MANUSCRIPT insipidus was 81% (specificity 97%); conversely, if levels increased to >30 pmol/L, the negative predictive value was 95% (sensitivity 94%). This shows that low copeptin levels despite surgical stress reflect postoperative diabetes inspidus, while high levels virtually exclude it. Copeptin may therefore become a novel tool for early goal-directed management of postoperative diabetes insipidus (Figure 3). Interestingly, in this study, copeptin values of patients with isolated syndrome of
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inappropriate ADH secretion (SIADH) showed similar changes pre-and postoperatively as patients with uneventful postoperative course. Thus on the basis of copeptin levels, no discrimination between postoperatively normal and elevated AVP activity was possible.
In patients presenting with polyuria/polydipsia, copeptin was evaluated as a biomarker for the
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differential diagnosis. A prospective study showed that copeptin was able to separate patients with complete nephrogenic and central diabetes insipidus by a single baseline measurement (29). The two patients with complete nephrogenic diabetes insipidus had
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baseline copeptin values greater than 20 pmol/L and the nine patients with complete central diabetes insipidus had baseline copeptin levels below 2.6 pmol/L. More difficult was the differentiation between patients with partial central diabetes insipidus and primary polydipsia. Here, a baseline cut-off value >3 pmol/L had a sensitivity and specificity of 82% and 92% to diagnose primary polydipsia, and a value >5 pmol/L in a state of dehydration revealed a sensitivity and specificity of 96% and 81% (29). Although this diagnostic yield is much better
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compared to the water deprivation test or direct AVP measurement, the overlap in single hormone levels still hampered the achievement of absolutely discriminating diagnostic results. The ratio of plasma copeptin increase during dehydration in relation to the serum sodium concentration measured at the end of the test was found to be a more successful
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parameter in this study. With this ratio,, a cutoff value >20 pmol/L/mmol/L (*1000) attained a diagnostic accuracy of 94%, with a specificity and sensitivity of 100% and 86%, to separate primary polydipsia from partial central diabetes insipidus.
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An important limitation of the standard water deprivation test, namely its limited sufficiency in producing hyperosmolar plasma levels, was taken into account in a most recent study of 55 patients with polyuria/polydipsia presenting to endocrine clinics of different tertiary care centers in Switzerland and Germany. In this study, it was shown that a modified water deprivation test followed by hypertonic saline infusion with copeptin measurement yielded reliably hyperosmolar sodium levels and consequently higher sensitivity and specificity values for plasma copeptin in the differential diagnosis of polyuria/polydipsia (40). All patients underwent a standardized water deprivation test starting at 8 AM, without prior fluid restriction. The test was stopped when plasma sodium exceeded 147 mmol/L. If plasma sodium levels by water deprivation alone increased to >147 mmol/L, and urine osmolality remained < 300 mmol/kg H2O, the test was discontinued and a desmopressin challenge was 8
ACCEPTED MANUSCRIPT performed. If plasma sodium did not exceed 147 mmol/L by thirsting alone by 1PM, patients received a 3% saline infusion and the test was terminated when plasma sodium exceeded 147 mmol/L. Without prior thirsting, a single baseline copeptin level >21.4 pmol/L differentiated nephrogenic diabetes inspidus from other etiologies with 100% sensitivity and specificity, rendering water deprivation testing unnecessary in these cases. A stimulated copeptin >4.9
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pmol/L (at sodium levels >147 mmol/L) differentiated between patients with primary polydipsia and patients with partial central diabetes insipidus with 94.0% specificity and 94.4% sensitivity; a stimulated AVP >1.8 pg/ml differentiated between these same categories with 93.0% specificity and 83.0% sensitivity. This study showed again that plasma copeptin
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is a reliable marker to discriminate between different entities of the polyuria/polydipsia and correlates well with AVP plasma levels (Figure 4a and b). A possible new algorithm for the differential diagnosis of PPS is given in Figure 5.
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Unfortunately, the water deprivation test remains a demanding and long test procedure and is associated with patient discomfort. Another important limitation of the water deprivation test is that volume depletion is a strong stimulus for AVP secretion and accordingly higher levels of AVP and copeptin must be expected after a water deprivation test compared to a volume-neutral osmotic stimulation. Therefore, an ideal test method for differentiation of the various etiologies of polyuria/polydipsia would be as resistant as possible against any non-
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osmotic AVP stimuli. This could, for example, be achieved by a hypertonic saline bolus injection. In fact, measuring copeptin responsivity to a hypertonic saline infusion without prior thirsting, could be a promising, more reliable, comfortable, and less time consuming test method than the standard procedure The performance of copeptin in the differential
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diagnosis of polyuria/polydipsia comparing the gold-standard water deprivation test against hypertonic saline infusion test with copeptin measurement is currently evaluated in a multicenter international study (NCT01940614). The objective of this study will be first to test
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whether the short hypertonic saline infusion test is able to replace the complicated water deprivation test as gold standard test method for osmotic stimulation. Secondary objectives will be to demonstrate that copeptin measurements can replace the classic AVP assay, and to generate qualified diagnostic copeptin cut-off values for differentiation between primary polydipsia and partial central diabetes insipidus based upon the hypertonic saline infusion test method. 3)
Copeptin as a biomarker in hyponatremia Hyponatremia is the most common fluid and electrolyte disorder (41). The prevalence of hyponatremia, defined as a serum sodium less than 135 mmol/liter, may be as high as 15– 30% in hospitalized patients. The prevalence of profound hyponatremia, defined as a serum 9
ACCEPTED MANUSCRIPT sodium value below 125 mmol/L, ranges between 2-3% per 100 hospitalized patients per day (42) and is known to be associated with higher morbidity, mortality and prolonged length-ofhospital stay. Hyponatremic patients have 5%–50% all-cause death rates (43), depending on comorbidities, and the condition is associated with more frequent falls, fractures and osteoporosis (44). Consequently incorrect differential diagnosis and suboptimal treatment may have devastating consequences and it is crucial to accurately identify the etiology
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underlying hyponatremia and begin effective therapy as early as possible. Using clinical signs and routine laboratory variables for the differential diagnosis of hyponatremia has shown limited sensitivity and specificity (<50%) (45). Much research interest has therefore focused on the evaluation of serum and urine parameters or clinical biomarkers to enhance
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the pathophysiological categorization and accelerate the initiation of appropriate sodium correction.
A prospective observational study including 106 patients with hyponatremia has shown that
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copeptin measurement reliably identified patients with primary polydipsia (n=4), but had limited utility in the differential diagnosis of other hyponatremic disorders. Specifically, copeptin levels in healthy volunteers were 4 pmol/L (25th to 75th percentile 2-6), 2 pmol/L (5th to 75th percentile 1-3) in primary polydipsia, 5 pmol/L (5th to 75th percentile 3-23) in diureticinduced hyponatremia, 10 pmol/L (5th to 75th percentile 4-21) in SIADH, 16 pmol/L (5th to 75th percentile 9-39) in sodium depleted patients and 23 pmol/L (5th to 75th percentile 10-50) in
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sodium expanded patients. In contrast, the copeptin to U-Na ratio differentiated accurately between volume-depleted and normovolemic disorders (area under the receiver-operating characteristic curve (ROC) 0.88, 95% confidence interval 0.81–0.95), resulting in a sensitivity and specificity of 85 and 87% if a cutoff value of 30 pmol/mmol was used. The combined
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information of a plasma copeptin less than 3 pmol/liter and a urine osmolality less than 200 mOsm/kg identified primary polydipsia in 100% of suspected patients (46) (Figure 6). A more recent study aimed at evaluating the reliability of copeptin measurements in
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assessing intravascular volume depletion in hypoosmolar hyponatremic patients. This study included 131 hospitalized patients with chronic severe hyponatremia and classified them as having decreased or as having normal/expanded intravascular volume. The results showed that copeptin levels were higher in patients with decreased intravascular volume compared to normal or expanded intravascular volume (34.6 vs. 11.3 pmol/L) and exhibited a reliable performance for assessment of decreased intravascular volume (ROC AUC at 0.717 [95% CI 0.629–0.805]). The combination of copeptin and Ur/Cr resulted in an improved ROC AUC up to 0.787 (95% CI 0.709–0.866). This suggests that copeptin can be used to reflect intravascular volume state and could be a biomarker for its assessment. Copeptin may, therefore, be a useful marker especially in case of confusing clinical presentation (47).
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ACCEPTED MANUSCRIPT Another still unpublished study confirms these previous results. In a prospective multicenter observational study in Switzerland, 298 patients admitted to the emergency department with profound hyponatremia were included. Copeptin levels were lowest in patients with primary polydipsia and a copeptin level <3.9 pmol/L identified primary polydipsia with a high specificity of 91%. Conversely, copeptin levels were highest in patients with hypovolemic hyponatremia and a copeptin level >84 pmol/L predicted hypovolemia with a specificity of
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90%. The copeptin/urinary sodium ratio in patients with SIADH was lower as compared to patients with other hyponatremic etiologies. However, the specificity to identify SIADH was only moderate (48).
In a retrospective secondary analysis involving 80 hyponatremic patients from a total of 1054
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inpatients with lower respiratory tract infection or acute cerebrovascular events, copeptin concentrations were significantly higher in hypervolemic patients and those with heart or kidney failure than in other hyponatremic patients (49). Copeptin did not distinguish between
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other hyponatremia etiologies. As mentioned above, copeptin accurately correlates with the severity of sepsis, ischemic stroke or heart failure. Therefore, in patients with concomitant serious underlying diseases or acute cerebrovascular events, but mild hyponatremia, stressinduced copeptin release may confound interpretation of copeptin levels based on hyponatremia-related homeostatic stimuli (49).
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Taken together, these studies demonstrate that low copeptin levels identify patients with primary polydipsia, whilst high copeptin levels rather point to hypovolemic conditions of hyponatremia. However, the diagnostic utility of copeptin is limited in other hyponatremic disorders and the stress commonly associated with underlying diseases present in these
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3) Summary
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patients must be taken into account in interpreting copeptin levels.
Copeptin is generated from pre-provasopressin and is secreted from the posterior pituitary in an equimolar ratio together with AVP and neurophysin II. It is a 39 amino acid glycosylated peptide and is released together with AVP during precursor processing. Although detected decades ago, it was not used as a biomarker until 2006 when a novel sandwich immunoassay was developed allowing its simple measurement. In the following years, copeptin was investigated as a biomarker in AVP-dependent diseases. In the evaluation of polyuria/polydipsia copeptin measurements show a high diagnostic accuracy in the differential diagnosis, with a single baseline level allowing to unequivocally identifying patients with complete nephrogenic (without prior thirsting) or complete central (after an overnight dehydration period) diabetes insipidus (29, 40). In the differentiation of mild forms 11
ACCEPTED MANUSCRIPT of diabetes insipidus from primary polydipsia, copeptin measured at a sodium level of >147 mmol/L had sensitivities and specificities of >94%. In patients undergoing pituitary surgery, copeptin predicts development of later diabetes insipidus and may be a biomarker improving early goal-directed management of postoperative diabetes insipidus. The diagnostic use of copeptin in hyponatremia is more complicated. Different prospective studies have investigated copeptin levels in mild to profound hyponatremia. Copeptin levels
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were shown to be lowest in patients with primary polydipsia and highest in hypovolemic hyponatremia. Copeptin also seems to mirror intravascular volume state. However, there is a wide overlap of copeptin levels in the various etiologies of hyponatremia prohibiting its widespread use as a biomarker in the differential diagnosis of hyponatremia. The copeptin to ratio
differentiates
more
accurately
between
volume-depleted
and
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urinary-sodium
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normovolemic disorders.
Practice points:
In patients with polyuria/polydipsia, baseline copeptin levels >20 pmol/L without prior fluid deprivation identify patients with nephrogenic diabetes insipidus; baseline copeptin levels <2.6 pmol/L with prior fluid deprivation identify patients with complete central diabetes insipidus
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In patients with polyuria/polydipsia, copeptin levels obtained after osmotic stimulation differentiate patients with partial central diabetes insipidus from patients with primary polydipsia with high sensitivities and specificities >94% In hyponatremia, low copeptin levels <4 pmol/L point to primary polydipsia, high levels of
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copeptin >80 pmol/L point to hypovolemic hyponatremia In other etiologies of hyponatremia, copeptin levels overlap widely, thereby limiting its use in
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the differential diagnosis
Research Agenda:
Establish half-life time of copeptin compared to AVP Validate proposed copeptin cutoff values for the differential diagnosis of polyuria/polydipsia in large prospective studies Establish the value of copeptin measurements after hypertonic saline infusion as a new standard test in the differential diagnosis of patients with polyuria/polydipsia.
Conflict of interest statement
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ACCEPTED MANUSCRIPT
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MCC and WF received speaker honoraria from Thermofisher AG, the manufacturer of the copeptin assay. NGM was once employed by Thermofisher, but has currently no conflict of interest.
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References
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1. Land H, Schutz G, Schmale H, et al. Nucleotide sequence of cloned cDNA encoding bovine arginine vasopressin-neurophysin II precursor. Nature. 1982;295(5847):299-303. 2. Levy B, Chauvet MT, Chauvet J, et al. Ontogeny of bovine neurohypophysial hormone precursors. II. Foetal copeptin, the third domain of the vasopressin precursor. International journal of peptide and protein research. 1986;27(3):320-4. 3*. Morgenthaler NG, Struck J, Alonso C, et al. Assay for the measurement of copeptin, a stable peptide derived from the precursor of vasopressin. Clin Chem. 2006;52(1):112-9. 4. Holwerda DA. A glycopeptide from the posterior lobe of pig pituitaries. I. Isolation and characterization. European journal of biochemistry / FEBS. 1972;28(3):334-9. 5. Nagy G, Mulchahey JJ, Smyth DG, et al. The glycopeptide moiety of vasopressin-neurophysin precursor is neurohypophysial prolactin releasing factor. Biochemical and biophysical research communications. 1988;151(1):524-9. 6. Hyde JF, North WG, Ben-Jonathan N. The vasopressin-associated glycopeptide is not a prolactin-releasing factor: studies with lactating Brattleboro rats. Endocrinology. 1989;125(1):35-40. 7. Barat C, Simpson L, Breslow E. Properties of human vasopressin precursor constructs: inefficient monomer folding in the absence of copeptin as a potential contributor to diabetes insipidus. Biochemistry. 2004;43(25):8191-203. 8. Parodi AJ. Protein glucosylation and its role in protein folding. Annual review of biochemistry. 2000;69:69-93. 9. Schrag JD, Procopio DO, Cygler M, et al. Lectin control of protein folding and sorting in the secretory pathway. Trends in biochemical sciences. 2003;28(1):49-57. 10*. Balanescu S, Kopp P, Gaskill MB, et al. Correlation of plasma copeptin and vasopressin concentrations in hypo-, iso-, and hyperosmolar States. The Journal of clinical endocrinology and metabolism. 2011;96(4):1046-52. 11. Morgenthaler N. Measurement of AVP and copeptin; in: copeptin – Biochemistry and Clinical Diagnostics. Uni-Med Verlag. 2014:16-20. 12. Bhandari SS, Loke I, Davies JE, et al. Gender and renal function influence plasma levels of copeptin in healthy individuals. Clinical science (London, England : 1979). 2009;116(3):257-63. 13. Darzy KH, Dixit KC, Shalet SM, et al. Circadian secretion pattern of copeptin, the C-terminal vasopressin precursor fragment. Clin Chem. 2010;56(7):1190-1. 14*. Szinnai G, Morgenthaler NG, Berneis K, et al. Changes in plasma copeptin, the c-terminal portion of arginine vasopressin during water deprivation and excess in healthy subjects. The Journal of clinical endocrinology and metabolism. 2007;92(10):3973-8. 15. Maeder MT, Staub D, Brutsche MH, et al. Copeptin response to clinical maximal exercise tests. Clin Chem. 2010;56(4):674-6. 13
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16. Hew-Butler T, Hoffman MD, Stuempfle KJ, et al. Changes in copeptin and bioactive vasopressin in runners with and without hyponatremia. Clinical journal of sport medicine : official journal of the Canadian Academy of Sport Medicine. 2011;21(3):211-7. 17. Walti C, Siegenthaler J, Christ-Crain M. Copeptin levels are independent of ingested nutrient type after standardised meal administration--the CoMEAL study. Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals. 2014;19(7):557-62. 18. Katan M, Fluri F, Morgenthaler NG, et al. Copeptin: a novel, independent prognostic marker in patients with ischemic stroke. Annals of neurology. 2009;66(6):799-808. 19. Reichlin T, Hochholzer W, Stelzig C, et al. Incremental value of copeptin for rapid rule out of acute myocardial infarction. Journal of the American College of Cardiology. 2009;54(1):60-8. 20. Katan M, Christ-Crain M. The stress hormone copeptin: a new prognostic biomarker in acute illness. Swiss medical weekly. 2010;140:w13101. 21. Siegenthaler J, Walti C, Urwyler SA, et al. Copeptin concentrations during psychological stress: the PsyCo study. European journal of endocrinology / European Federation of Endocrine Societies. 2014;171(6):737-42. 22. Urwyler SA, Schuetz P, Sailer C, et al. Copeptin as a stress marker prior and after a written examination - the CoEXAM study. Stress. 2015:1-4. 23*. Fenske W, Allolio B. Clinical review: Current state and future perspectives in the diagnosis of diabetes insipidus: a clinical review. The Journal of clinical endocrinology and metabolism. 2012;97(10):3426-37. 24. Carter AC, Robbins J. The use of hypertonic saline infusions in the differential diagnosis of diabetes insipidus and psychogenic polydipsia. The Journal of clinical endocrinology and metabolism. 1947;7(11):753-66. 25. Barlow ED, De Wardener HE. Compulsive water drinking. Q J Med. 1959;28(110):235-58. 26. Dies F, Rangel S, Rivera A. Differential diagnosis between diabetes insipidus and compulsive polydipsia. Annals of internal medicine. 1961;54:710-25. 27. Dashe AM, Cramm RE, Crist CA, et al. A water deprivation test for the differential diagnosis of polyuria. Jama. 1963;185:699-703. 28. Miller M, Dalakos T, Moses AM, et al. Recognition of partial defects in antidiuretic hormone secretion. Annals of internal medicine. 1970;73(5):721-9. 29*. Fenske W, Quinkler M, Lorenz D, et al. Copeptin in the differential diagnosis of the polydipsiapolyuria syndrome--revisiting the direct and indirect water deprivation tests. The Journal of clinical endocrinology and metabolism. 2011;96(5):1506-15. 30. Robertson GL. Diabetes insipidus. Endocrinology and metabolism clinics of North America. 1995;24(3):549-72. 31. Czernichow A, Robinson A. Diabetes insipidus in man. Karger. 1985:176. 32. Milles JJ, Spruce B, Baylis PH. A comparison of diagnostic methods to differentiate diabetes insipidus from primary polyuria: a review of 21 patients. Acta Endocrinol (Copenh). 1983;104(4):4106. 33. Zerbe RL, Robertson GL. A comparison of plasma vasopressin measurements with a standard indirect test in the differential diagnosis of polyuria. N Engl J Med. 1981;305(26):1539-46. 34. Baylis PH. Diabetes insipidus. Journal of the Royal College of Physicians of London. 1998;32(2):108-11. 35. Baylis PH, Gaskill MB, Robertson GL. Vasopressin secretion in primary polydipsia and cranial diabetes insipidus. Q J Med. 1981;50(199):345-58. 36. Robertson GL, Mahr EA, Athar S, et al. Development and clinical application of a new method for the radioimmunoassay of arginine vasopressin in human plasma. J Clin Invest. 1973;52(9):234052. 37. Czaczkes JW, Kleeman CR, Koenig M. PHYSIOLOGIC STUDIES OF ANTIDIURETIC HORMONE BY ITS DIRECT MEASUREMENT IN HUMAN PLASMA. J Clin Invest. 1964;43:1625-40.
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38*. Katan M, Morgenthaler NG, Dixit KC, et al. Anterior and posterior pituitary function testing with simultaneous insulin tolerance test and a novel copeptin assay. The Journal of clinical endocrinology and metabolism. 2007;92(7):2640-3. 39*. Winzeler B, Zweifel C, Nigro N, et al. Postoperative copeptin concentration predicts diabetes insipidus after pituitary surgery. The Journal of clinical endocrinology and metabolism. 2015:jc20144527. 40*. Timper K, Fenske W, Kuhn F, et al. Diagnostic accuracy of copeptin in the differential diagnosis of the polyuria-polydipsia syndrome: A prospective multicenter study. The Journal of clinical endocrinology and metabolism. 2015:jc20144507. 41. Kumar S, Berl T. Sodium. Lancet. 1998;352(9123):220-8. 42. Natkunam A, Shek CC, Swaminathan R. Hyponatremia in a hospital population. Journal of medicine. 1991;22(2):83-96. 43. Upadhyay A, Jaber BL, Madias NE. Incidence and prevalence of hyponatremia. The American journal of medicine. 2006;119(7 Suppl 1):S30-5. 44. Verbalis JG, Barsony J, Sugimura Y, et al. Hyponatremia-induced osteoporosis. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 2010;25(3):554-63. 45. Chung HM, Kluge R, Schrier RW, et al. Clinical assessment of extracellular fluid volume in hyponatremia. The American journal of medicine. 1987;83(5):905-8. 46*. Fenske W, Stork S, Blechschmidt A, et al. Copeptin in the differential diagnosis of hyponatremia. The Journal of clinical endocrinology and metabolism. 2009;94(1):123-9. 47. Boursier G, Almeras M, Buthiau D, et al. CT-pro-AVP as a tool for assessment of intravascular volume depletion in severe hyponatremia. Clinical biochemistry. 2015. 48. Nigro N, Winzeler B, Suter I, et al. Copeptin in the differential diagnosis and therapy management of hyponatremia in hospitalized patients-’’The Co-MED-Study’’. Endocrine Society Meeting 2014, Chicago. 2014. 49. Nigro N, Muller B, Morgenthaler N, et al. The use of copeptin, the stable peptide of the vasopressin precursor, in the differential diagnosis of sodium imbalance in patients with acute diseases. Swiss medical weekly. 2011;141:w13270. 50. Morgenthaler N. AVP and copeptin: relationship and function, in: copeptin – Biochemistry and Clinical Diagnostics. Uni-Med Verlag. 2014:16-20. 51. Morgenthaler N. AVP and copeptin: relationship and function, in: copeptin – Biochemistry and Clinical Diagnostics. Uni-Med Verlag. 2014:28-31.
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Legends for the figures
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Figure 1 Pre Pro-Arginine Vasopressin (AVP)-Neurophysin precursor peptide, which is processed to generate a signal peptide, AVP, neurophysin II and the carboxy-terminal glycopeptide copeptin (from (50)). Figure 2 Correlation of Arginine Vasopressin (AVP) and copeptin with plasma osmolality. Plasma AVP and copeptin concentrations measured during water load and hypertonic saline tests are shown as scatter plot. rS denotes Spearman’s rank correlation coefficients (from (51)).
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Figure 3 Initial postoperative copeptin levels to predict development of diabetes insipidus after pituitary surgery. Dark grey bars represent all patients (n=205), light grey bars represent only patients where copeptin was measured within the first 12 hours postoperatively (n=157) (modified from (39)).
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Figure 4a Baseline AVP and copeptin plasma levels in the differential diagnosis of the polyuria/polydipsia Box plots depicting interquartile ranges with medians and whiskers depicting minimal and maximal values for baseline AVP (A) and copeptin (B) levels without prior thirsting in patients with complete and partial central diabetes insipidus, patients with primary polydipsia, and patients with complete and partial nephrogenic diabetes insipidus. Cut-offs for best discrimination between nephrogenic versus non-nephrogenic diabetes insipidus for AVP and copeptin are shown. The overall P value refers to Kruskal-Wallis test results across all patient subgroups. For all two-subgroup comparisons, Mann-Whitney tests were used and the P value was Bonferroni-adjusted. AVP = arginine vasopressin; DI = diabetes insipidus; PP = primary polydipsia (modified from (40)). Figure 4b Osmotically-stimulated AVP and copeptin plasma levels in the differential diagnosis of polyuria/polydipsia Box and whisker plots with medians and minimal and maximal values for stimulated AVP (A) and copeptin (B) plasma values at a plasma sodium level >147 mmol/L are depicted for patients with complete and partial central diabetes insipidus and for patients with primary polydipsia. Osmotic stimulation was provided by a combined water deprivation and saline infusion test. The cut-offs for best discrimination between primary polydipsia versus central diabetes insipidus for AVP and copeptin are depicted. The overall P value refers to Kruskal-Wallis test results across all patient subgroups. For all two-subgroup comparisons, Mann-Whitney tests were used and the P value was 16
ACCEPTED MANUSCRIPT Bonferroni-adjusted. AVP = arginine vasopressin; DI = diabetes insipidus; PP = primary polydipsia (modified from(40)). Figure 5 Proposed new algorithm for the differential diagnosis of patients with polyuria/polydipsia
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Figure 6 Copeptin in the differential diagnosis of hyponatremia. Box and whisker plots for the copeptin to U-Na+ ratio in five diagnostic groups of hyponatremic patients (n=106) and healthy controls (n=32). *, P <0.05 compared with control (ANOVA with Holm‘s post hoc test); ª, P < 0.05 compared with SIADH (ANOVA with Holm‘s post hoc test) (modified from(46)).
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S1521-690X(16)00008-7
DOI:
10.1016/j.beem.2016.02.003
Reference:
YBEEM 1077
To appear in:
Best Practice & Research Clinical Endocrinology & Metabolism
Please cite this article as: Christ-Crain M, Morgenthaler NG, Fenske PW, Copeptin as a biomarker and a diagnostic tool in the evaluation of patients with polyuria-polydipsia and hyponatremia, Best Practice & Research Clinical Endocrinology & Metabolism (2016), doi: 10.1016/j.beem.2016.02.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title Page
Copeptin as a biomarker and a diagnostic tool in the evaluation of patients with polyuria-polydipsia and hyponatremia
Prof. M. Christ-Crain, MD, PhD (corresponding author)
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Authors:
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Department of Endocrinology, University hospital Basel, University of Basel, Switzerland Email: [email protected]; Tel. +41 61 328 7080 Fax. +41 61 265 5100
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N. G. Morgenthaler, MD, PhD Institut für Experimentelle Endokrinologie, Charité-Universitätsmedizin-Berlin , Berlin, Germany Email: [email protected] Tel. +49 42122051610
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PD W. Fenske, MD, PhD
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Leipzig University Medical Center, Integrated Research and Treatment Center for Adiposity Diseases Email: [email protected] Tel: +49 341 9713306
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ACCEPTED MANUSCRIPT Abstract Copeptin is part of the 164 amino acid precursor protein preprovasopressin together with vasopressin and neurophysin II. During precursor processing, copeptin is released together with vasopressin. Copeptin concentrations respond as rapidly as vasopressin to changes in
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osmolality, a decrease in blood pressure or stress and there is a close correlation of vasopressin and copeptin concentrations. For these reasons, copeptin is propagated as a surrogate marker for vasopressin in the differential diagnosis of the polyuria-polydipsia
syndromes and hyponatremia. Results of prospective studies show that a baseline copeptin level without prior fluid deprivation >20 pmol/L is able to identify patients with nephrogenic
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diabetes insipidus, whereas osmotically stimulated copeptin levels differentiate between patients with partial central diabetes insipidus and primary polydipsia with a high sensitivity
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and specificity >94%. In hyponatremia, low copeptin levels point to primary polydipsia and high levels to hypovolemic hyponatremia. The copeptin to urinary sodium ratio differentiates accurately between volume-depleted and normovolemic disorders.
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Key words: Arginine Vasopressin, copeptin, polyuria-polydipsia syndrome, diabetes insipidus, hyponatremia
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ACCEPTED MANUSCRIPT Background: Copeptin Synthesis and function of copeptin Copeptin is part of the 164 amino acid precursor protein preprovasopressin together with vasopressin (AVP) and neurophysin II. It is a glycosylated peptide of 39 amino acids with a leucine-rich core region (1, 2). During precursor processing, copeptin is released together
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with AVP from magno- and parvocellular neurons of the hypothalamus. The molecular mass of copeptin is around 5 kDa which is in accordance with the theoretical prediction of processed copeptin (3) (Figure 1).
In 1972, Holwerda first described a 39 amino acid glycopeptide from the posterior pituitary of
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pigs (4). Levy et al. later termed this glycopeptide “copeptin” (2). It remained relatively unknown except to a few experts in the field of diagnostics until it was characterized in 2006 as a potential biomarker (3). Since the development of the first copeptin assay, the number
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of original publications on copeptin increases every year almost exponentially. In contrast to AVP, the physiological function of copeptin is still not fully understood. The other precursor fragment neurophysin II acts as a carrier protein in the transport of AVP from the hypothalamus to the neurohypophysis, and some experiments suggested the role of copeptin as a prolactin-releasing factor, albeit with inconclusive results (5, 6). Other work indicates that copeptin may play a role in the correct structural formation of proAVP (7).
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Reports on the interaction with the calnexin/ calreticulin system (8, 9), which monitors protein folding and interacts with glycosylated proteins, suggest a function as a chaperone, decreasing the formation of inactive, and increasing the formation of active hormones. It is tempting to attribute the inefficient monomer folding in the absence of copeptin to the
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pathogenesis of central diabetes insipidus, but this needs further examination. Also, it does not explain why oxytocin, the other neurohormone with similar structure to AVP, does not require a copeptin-like carboxy-terminal fragment for correct maturation.
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Copeptin response to osmotic and fluid status in healthy volunteers It is known that the copeptin concentration rises as rapidly as AVP to changes in osmolality, a decrease in blood pressure or to unspecific stress. This increase can be explained by the equimolar production together with AVP. The fact that copeptin is stable in serum or plasma for days, once the blood sample is removed from the body, indicates that copeptin is either processed by tissue-bound proteases, and/or rapidly eliminated via the kidneys or the liver. Copeptin does not accumulate in the body as an irrelevant side product of AVP synthesis, but no specific copeptin receptor or copeptin elimination mechanism has been identified. Evidence for a partial renal elimination is provided by copeptin measurements in the urine.
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ACCEPTED MANUSCRIPT A direct comparison between copeptin and AVP serum concentrations in relationship to serum osmolality was done in collaboration with a group of AVP experts (10) (Figure 2). In this study, plasma osmolality, copeptin, and AVP levels were measured in 20 volunteers at baseline, after an oral water load, and during and after i.v. infusion of 3% saline. During the experiment, median plasma osmolality decreased from 290 mOsm/kg (range, 284–302) at baseline to 281 (273–288) mOsm/kg after water load and rose to 301 (298–307) mOsm/kg
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after hypertonic saline. At the same time, median plasma copeptin concentrations decreased from 3.3 (1.1–36.4) pmol/L at baseline to 2.0 (0.9 –10.4) pmol/L after water load and increased to 13.6 (3.7– 43.3) pmol/L after hypertonic saline. The correlation between copeptin and serum osmolality was stronger (Spearman’s rank correlation coefficient r=0.77)
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than between AVP and serum osmolality (r=0.49). There was a close correlation between AVP and copeptin concentrations (r=0.8). The strength of the correlation between AVP and copeptin depends on the quality of the AVP assay used, but most data show a correlation as
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would be expected from the equimolar release of both peptides (3).
Technical Advantages of Copeptin Measurement compared to AVP Measurement of mature AVP is difficult due to its small molecular size and subject to preanalytical errors. The small size prevents the efficient binding of two antibodies at the same time, which would be needed for sensitive sandwich immunoassays. Preanalytical errors
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include the strong binding of AVP to thrombocytes in the serum (due to the presence of vasopressin-receptors), and the short half-life time in vivo (3, 11). In contrast, for copeptin two assays are available with sufficient technical description and clinical data justifying their routine clinical use. These are the original sandwich immunoluminometric assay (LIA) (3) and
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its automated immunofluorescent successor (on the KRYPTOR platform). For both assays, CE IVD (European Community In Vitro Diagnostics) approval is available, but FDA (United States Federal Drug Administration) approval is still pending.
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The advantages of copeptin measurement compared to AVP mainly involve (i) the lower minimally required volume of only 50 µL serum or plasma, (ii) no
requirements for an
extraction step or other pre-analytical procedures such as the addition of protease inhibitors, (iii) results are in general available in approximately 0.5 to 2.5 hours, and (iv) as a sandwich immunoassay, it is more sensitive than competitive AVP immunoassays; (v) finally, copeptin, unlike mature AVP, is much more stable in plasma or serum ex vivo. Ex vivo stability of copeptin (<20% loss of recovery) was shown for serum and plasma for at least seven days at room temperature and at 14 days at 4°C.
Normal values and physiological response of plasma copeptin in healthy volunteers
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ACCEPTED MANUSCRIPT The reference range for copeptin was first defined in a study with 359 healthy volunteers. This study showed the broad range of copeptin values. The median copeptin plasma concentration was 4.2 pmol/L with a range between 1- 13.8 pmol/L (3). Similarly, copeptin concentrations in healthy volunteers of a population of 5000 individuals ranged between 1 and 13 pmol/L (upper 97.5 percentile) with median values <5 pmol/L (12). Men consistently show slightly higher values than women, but the difference in median values is only about 1
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pmol/L (12). Copeptin plasma concentration shows no correlation with age (3). The circadian rhythm of copeptin has only been investigated in a small study in 7 healthy volunteers, showing no consistent circadian rhythm (13).
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It is important to note that copeptin may fluctuate according to individual physiological conditions. In a study of 24 healthy volunteers, water deprivation over 28 hours increased serum copeptin from 4.6±1.7 pmol/L to 9.2±5.2 pmol/L (p<0.001). Additional infusion of
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hypertonic saline further increased plasma copeptin from 4.9±3.0 pmol/L to 19.9±4.8 pmol/L (p<0.001). Conversely, copeptin decreased from 6.2±2.4 pmol/L to 2.4±2.1 pmol/L (p<0.01) during hypotonic saline infusion (14). Essentially, copeptin showed identical changes during disordered water states or osmolality as previously shown for AVP.
In response to exercise, the copeptin concentration in blood increases, but does not exceed
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the 99th percentile of the reference range (15, 16).
In contrast, copeptin levels seem not to be affected by food intake: in a study in 30 healthy volunteers undergoing an oral glucose tolerance test and a mixed meal tolerance test, median copeptin levels remained unchanged or even decreased slightly (from 4.9 [3.6–8.3]
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and 4.9 [3.6–7.1] pmol/l to 3.2 (2.8–5.9) and 4.1 (2.7–6.1) pmol/l after the respective tests). This suggests that copeptin levels can be interpreted independently of the prandial status (17). In contrast, already small amounts of fluid (200-300 ml) seem to significantly decrease
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copeptin levels, underlining that any recent fluid intake must be considered when interpreting copeptin levels (17).
Copeptin response to unspecific stressors As for AVP, the main stimulus for copeptin is an increase in osmolality or a decrease in plasma volume. In addition, somatic stress, e.g. due to disease, is a major determinant of copeptin levels. Copeptin was shown to more subtly mirror the individual stress level as compared to cortisol. Accordingly, copeptin increases with increasing severity of disease in pneumonia, ischemic stroke and other diseases (18-20). Due to this positive association of copeptin with the severity of illness, copeptin has been proposed as a prognostic marker in acute illness. As such, copeptin has been analyzed in sepsis, pneumonia, lower respiratory 5
ACCEPTED MANUSCRIPT tract infections, stroke and other acute illnesses and was found to discriminate patients with unfavorable outcomes from patients with favourable outcomes (18-20). Moreover, copeptin improves the prognostic information provided by commonly used clinical scoring instruments. An accurate prognostic assessment has the potential to guide interventions and effectively plan and monitor rehabilitation and, thus optimize the management of individual patients and
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the allocation of limited health care resources.
In contrast to stress associated with somatic disease little is known about the influence of psychological stress on copeptin levels. Two recent studies in healthy volunteers undergoing a trier social stress test (TSST), and in medical students before and after a written medical
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exam, respectively have shown that copeptin increases (to a median of around 5 pmol/L, baseline 3.7 pmol/L) upon psychological stress, to a much smaller degree than observed in response to somatic disease (21, 22). This points to the fact that psychological stress has to
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be taken into account as a confounding factor in disorders with only subtly abnormal copeptin levels, e.g. in polyuria-polydipsia syndromes. However, in acute conditions like pneumonia or acute myocardial infarction, where copeptin levels often exceed the upper limit of the normal distribution, the modest increase seen in psychological stress is of lesser relevance. In contrast to cortisol, copeptin did not seem to predict examination performance in medical students (22).
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Interestingly, patients with complete central diabetes insipidus showed no increase upon psychological stress (peak 2.15 pmol/L (1.5–2.28)) in contrast to healthy volunteers, where copeptin levels increased significantly to 5.1 (3.2–7.0) pmol/L. By contrast, cortisol values were similar in patients and volunteers. This shows that the copeptin response to stress is
Copeptin as a biomarker in polyuria-polydipsia syndromes
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blunted in patients with diabetes insipidus (20).
The fact that copeptin mirrors AVP concentrations in the circulation predestines it as a biomarker in AVP-dependent diseases, especially in polyuria-polydipsia syndromes and hyponatremia. In the following, this review article will focus on copeptin as a diagnostic biomarker in these two specific entities. A spectrum of diseases like central diabetes insipidus, nephrogenic diabetes insipidus and primary polydipsia clinically present with polyuria and polydipsia (23). Careful differentiation of the underlying disorders is mandatory as treatment strategies vary substantially. After ruling out other, AVP-independent etiologies, such as hyperglycemia and hypercalcemia, the precise differentiation is often difficult to achieve (24). The available tests are not satisfactory (25-28) and often result in false diagnosis, especially in patients with 6
ACCEPTED MANUSCRIPT primary polydipsia and mild forms of diabetes insipidus (29-32). The widely accepted “goldstandard” in the differential diagnosis of polyuria/polydipsia is the water deprivation test with indirect assessment of the AVP activity by measurement of the maximally reached urine osmolality during a prolonged dehydration period or with direct measurement of AVP. However, recent findings revealed dramatic limitations of this procedure with an overall diagnostic accuracy of 70%, and an accuracy of only 41% in patients with primary polydipsia
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(29). Several reasons may account for this limited diagnostic outcome. Firstly, an accurate definition of the area defining the normal physiological relationship between plasma AVP and serum osmolality as an important prerequisite for the test interpretation, has long been missed (33-35), and accordingly, the biological variation of osmotic regulation and the non-
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linear association between plasma AVP and serum osmolality have not been accurately defined (29). Secondly, as mentioned above, the AVP assay per se is subject to several technical limitations, resulting in a high pre-analytical instability (3, 36, 37).
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Therefore, the availability of a novel immunoassay to measure copeptin has led to investigations whether measurement of copeptin can improve the differential diagnosis in patients with polyuria/polydipsia.
A first study in 2007 showed that hypoglycemia-induced copeptin response is a highly useful measure to identify patients with complete diabetes insipidus following transphenoidal pituitary surgery (38). In this study, patients with intact posterior pituitary function had basal
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copeptin levels of 3.7 ± 1.5 pmol/L with a maximal increase to 11.1 ± 4.6 pmol/L at 45 minutes after insulin injection. Copeptin levels in patients with diabetes insipidus were 2.4 ± 0.5 pmol/L before insulin injection with a maximum increase to 3.7 ± 0.7 pmol/L. Both basal and stimulated copeptin levels were lower in patients with central diabetes insipidus as
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compared to patients with intact posterior pituitary function. A stimulated copeptin level 45 minutes after insulin injection of less than 4.75 pmol/L had an optimal diagnostic accuracy to detect diabetes insipidus.
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Most recently, in the same population of patients undergoing pituitary surgery, copeptin levels measured at the day of surgery were shown to predict later diabetes insipidus (39). This study included 205 patients undergoing pituitary surgery at three Swiss or Canadian referral centers. Copeptin was measured pre-operatively and on each postoperative day until discharge. Fifty (24.4%) patients developed postoperative diabetes insipidus. Post-surgically, median copeptin levels in patients developing diabetes insipidus did not increase, and were lower than in patients without diabetes insipidus: 2.9 pmol/L vs 10.8 pmol/L, p<0.001. Logistic regression analysis showed a strong association of postoperative copeptin concentrations and diabetes insipidus, even after considering known predisposing factors for this complication (adjusted odds ratio (95% confidence interval) 1.41 (1.16–1.73). In patients with postoperative copeptin values <2.5 pmol/L, the positive predictive value for diabetes 7
ACCEPTED MANUSCRIPT insipidus was 81% (specificity 97%); conversely, if levels increased to >30 pmol/L, the negative predictive value was 95% (sensitivity 94%). This shows that low copeptin levels despite surgical stress reflect postoperative diabetes inspidus, while high levels virtually exclude it. Copeptin may therefore become a novel tool for early goal-directed management of postoperative diabetes insipidus (Figure 3). Interestingly, in this study, copeptin values of patients with isolated syndrome of
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inappropriate ADH secretion (SIADH) showed similar changes pre-and postoperatively as patients with uneventful postoperative course. Thus on the basis of copeptin levels, no discrimination between postoperatively normal and elevated AVP activity was possible.
In patients presenting with polyuria/polydipsia, copeptin was evaluated as a biomarker for the
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differential diagnosis. A prospective study showed that copeptin was able to separate patients with complete nephrogenic and central diabetes insipidus by a single baseline measurement (29). The two patients with complete nephrogenic diabetes insipidus had
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baseline copeptin values greater than 20 pmol/L and the nine patients with complete central diabetes insipidus had baseline copeptin levels below 2.6 pmol/L. More difficult was the differentiation between patients with partial central diabetes insipidus and primary polydipsia. Here, a baseline cut-off value >3 pmol/L had a sensitivity and specificity of 82% and 92% to diagnose primary polydipsia, and a value >5 pmol/L in a state of dehydration revealed a sensitivity and specificity of 96% and 81% (29). Although this diagnostic yield is much better
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compared to the water deprivation test or direct AVP measurement, the overlap in single hormone levels still hampered the achievement of absolutely discriminating diagnostic results. The ratio of plasma copeptin increase during dehydration in relation to the serum sodium concentration measured at the end of the test was found to be a more successful
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parameter in this study. With this ratio,, a cutoff value >20 pmol/L/mmol/L (*1000) attained a diagnostic accuracy of 94%, with a specificity and sensitivity of 100% and 86%, to separate primary polydipsia from partial central diabetes insipidus.
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An important limitation of the standard water deprivation test, namely its limited sufficiency in producing hyperosmolar plasma levels, was taken into account in a most recent study of 55 patients with polyuria/polydipsia presenting to endocrine clinics of different tertiary care centers in Switzerland and Germany. In this study, it was shown that a modified water deprivation test followed by hypertonic saline infusion with copeptin measurement yielded reliably hyperosmolar sodium levels and consequently higher sensitivity and specificity values for plasma copeptin in the differential diagnosis of polyuria/polydipsia (40). All patients underwent a standardized water deprivation test starting at 8 AM, without prior fluid restriction. The test was stopped when plasma sodium exceeded 147 mmol/L. If plasma sodium levels by water deprivation alone increased to >147 mmol/L, and urine osmolality remained < 300 mmol/kg H2O, the test was discontinued and a desmopressin challenge was 8
ACCEPTED MANUSCRIPT performed. If plasma sodium did not exceed 147 mmol/L by thirsting alone by 1PM, patients received a 3% saline infusion and the test was terminated when plasma sodium exceeded 147 mmol/L. Without prior thirsting, a single baseline copeptin level >21.4 pmol/L differentiated nephrogenic diabetes inspidus from other etiologies with 100% sensitivity and specificity, rendering water deprivation testing unnecessary in these cases. A stimulated copeptin >4.9
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pmol/L (at sodium levels >147 mmol/L) differentiated between patients with primary polydipsia and patients with partial central diabetes insipidus with 94.0% specificity and 94.4% sensitivity; a stimulated AVP >1.8 pg/ml differentiated between these same categories with 93.0% specificity and 83.0% sensitivity. This study showed again that plasma copeptin
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is a reliable marker to discriminate between different entities of the polyuria/polydipsia and correlates well with AVP plasma levels (Figure 4a and b). A possible new algorithm for the differential diagnosis of PPS is given in Figure 5.
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Unfortunately, the water deprivation test remains a demanding and long test procedure and is associated with patient discomfort. Another important limitation of the water deprivation test is that volume depletion is a strong stimulus for AVP secretion and accordingly higher levels of AVP and copeptin must be expected after a water deprivation test compared to a volume-neutral osmotic stimulation. Therefore, an ideal test method for differentiation of the various etiologies of polyuria/polydipsia would be as resistant as possible against any non-
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osmotic AVP stimuli. This could, for example, be achieved by a hypertonic saline bolus injection. In fact, measuring copeptin responsivity to a hypertonic saline infusion without prior thirsting, could be a promising, more reliable, comfortable, and less time consuming test method than the standard procedure The performance of copeptin in the differential
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diagnosis of polyuria/polydipsia comparing the gold-standard water deprivation test against hypertonic saline infusion test with copeptin measurement is currently evaluated in a multicenter international study (NCT01940614). The objective of this study will be first to test
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whether the short hypertonic saline infusion test is able to replace the complicated water deprivation test as gold standard test method for osmotic stimulation. Secondary objectives will be to demonstrate that copeptin measurements can replace the classic AVP assay, and to generate qualified diagnostic copeptin cut-off values for differentiation between primary polydipsia and partial central diabetes insipidus based upon the hypertonic saline infusion test method. 3)
Copeptin as a biomarker in hyponatremia Hyponatremia is the most common fluid and electrolyte disorder (41). The prevalence of hyponatremia, defined as a serum sodium less than 135 mmol/liter, may be as high as 15– 30% in hospitalized patients. The prevalence of profound hyponatremia, defined as a serum 9
ACCEPTED MANUSCRIPT sodium value below 125 mmol/L, ranges between 2-3% per 100 hospitalized patients per day (42) and is known to be associated with higher morbidity, mortality and prolonged length-ofhospital stay. Hyponatremic patients have 5%–50% all-cause death rates (43), depending on comorbidities, and the condition is associated with more frequent falls, fractures and osteoporosis (44). Consequently incorrect differential diagnosis and suboptimal treatment may have devastating consequences and it is crucial to accurately identify the etiology
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underlying hyponatremia and begin effective therapy as early as possible. Using clinical signs and routine laboratory variables for the differential diagnosis of hyponatremia has shown limited sensitivity and specificity (<50%) (45). Much research interest has therefore focused on the evaluation of serum and urine parameters or clinical biomarkers to enhance
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the pathophysiological categorization and accelerate the initiation of appropriate sodium correction.
A prospective observational study including 106 patients with hyponatremia has shown that
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copeptin measurement reliably identified patients with primary polydipsia (n=4), but had limited utility in the differential diagnosis of other hyponatremic disorders. Specifically, copeptin levels in healthy volunteers were 4 pmol/L (25th to 75th percentile 2-6), 2 pmol/L (5th to 75th percentile 1-3) in primary polydipsia, 5 pmol/L (5th to 75th percentile 3-23) in diureticinduced hyponatremia, 10 pmol/L (5th to 75th percentile 4-21) in SIADH, 16 pmol/L (5th to 75th percentile 9-39) in sodium depleted patients and 23 pmol/L (5th to 75th percentile 10-50) in
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sodium expanded patients. In contrast, the copeptin to U-Na ratio differentiated accurately between volume-depleted and normovolemic disorders (area under the receiver-operating characteristic curve (ROC) 0.88, 95% confidence interval 0.81–0.95), resulting in a sensitivity and specificity of 85 and 87% if a cutoff value of 30 pmol/mmol was used. The combined
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information of a plasma copeptin less than 3 pmol/liter and a urine osmolality less than 200 mOsm/kg identified primary polydipsia in 100% of suspected patients (46) (Figure 6). A more recent study aimed at evaluating the reliability of copeptin measurements in
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assessing intravascular volume depletion in hypoosmolar hyponatremic patients. This study included 131 hospitalized patients with chronic severe hyponatremia and classified them as having decreased or as having normal/expanded intravascular volume. The results showed that copeptin levels were higher in patients with decreased intravascular volume compared to normal or expanded intravascular volume (34.6 vs. 11.3 pmol/L) and exhibited a reliable performance for assessment of decreased intravascular volume (ROC AUC at 0.717 [95% CI 0.629–0.805]). The combination of copeptin and Ur/Cr resulted in an improved ROC AUC up to 0.787 (95% CI 0.709–0.866). This suggests that copeptin can be used to reflect intravascular volume state and could be a biomarker for its assessment. Copeptin may, therefore, be a useful marker especially in case of confusing clinical presentation (47).
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ACCEPTED MANUSCRIPT Another still unpublished study confirms these previous results. In a prospective multicenter observational study in Switzerland, 298 patients admitted to the emergency department with profound hyponatremia were included. Copeptin levels were lowest in patients with primary polydipsia and a copeptin level <3.9 pmol/L identified primary polydipsia with a high specificity of 91%. Conversely, copeptin levels were highest in patients with hypovolemic hyponatremia and a copeptin level >84 pmol/L predicted hypovolemia with a specificity of
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90%. The copeptin/urinary sodium ratio in patients with SIADH was lower as compared to patients with other hyponatremic etiologies. However, the specificity to identify SIADH was only moderate (48).
In a retrospective secondary analysis involving 80 hyponatremic patients from a total of 1054
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inpatients with lower respiratory tract infection or acute cerebrovascular events, copeptin concentrations were significantly higher in hypervolemic patients and those with heart or kidney failure than in other hyponatremic patients (49). Copeptin did not distinguish between
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other hyponatremia etiologies. As mentioned above, copeptin accurately correlates with the severity of sepsis, ischemic stroke or heart failure. Therefore, in patients with concomitant serious underlying diseases or acute cerebrovascular events, but mild hyponatremia, stressinduced copeptin release may confound interpretation of copeptin levels based on hyponatremia-related homeostatic stimuli (49).
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Taken together, these studies demonstrate that low copeptin levels identify patients with primary polydipsia, whilst high copeptin levels rather point to hypovolemic conditions of hyponatremia. However, the diagnostic utility of copeptin is limited in other hyponatremic disorders and the stress commonly associated with underlying diseases present in these
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3) Summary
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patients must be taken into account in interpreting copeptin levels.
Copeptin is generated from pre-provasopressin and is secreted from the posterior pituitary in an equimolar ratio together with AVP and neurophysin II. It is a 39 amino acid glycosylated peptide and is released together with AVP during precursor processing. Although detected decades ago, it was not used as a biomarker until 2006 when a novel sandwich immunoassay was developed allowing its simple measurement. In the following years, copeptin was investigated as a biomarker in AVP-dependent diseases. In the evaluation of polyuria/polydipsia copeptin measurements show a high diagnostic accuracy in the differential diagnosis, with a single baseline level allowing to unequivocally identifying patients with complete nephrogenic (without prior thirsting) or complete central (after an overnight dehydration period) diabetes insipidus (29, 40). In the differentiation of mild forms 11
ACCEPTED MANUSCRIPT of diabetes insipidus from primary polydipsia, copeptin measured at a sodium level of >147 mmol/L had sensitivities and specificities of >94%. In patients undergoing pituitary surgery, copeptin predicts development of later diabetes insipidus and may be a biomarker improving early goal-directed management of postoperative diabetes insipidus. The diagnostic use of copeptin in hyponatremia is more complicated. Different prospective studies have investigated copeptin levels in mild to profound hyponatremia. Copeptin levels
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were shown to be lowest in patients with primary polydipsia and highest in hypovolemic hyponatremia. Copeptin also seems to mirror intravascular volume state. However, there is a wide overlap of copeptin levels in the various etiologies of hyponatremia prohibiting its widespread use as a biomarker in the differential diagnosis of hyponatremia. The copeptin to ratio
differentiates
more
accurately
between
volume-depleted
and
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urinary-sodium
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normovolemic disorders.
Practice points:
In patients with polyuria/polydipsia, baseline copeptin levels >20 pmol/L without prior fluid deprivation identify patients with nephrogenic diabetes insipidus; baseline copeptin levels <2.6 pmol/L with prior fluid deprivation identify patients with complete central diabetes insipidus
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In patients with polyuria/polydipsia, copeptin levels obtained after osmotic stimulation differentiate patients with partial central diabetes insipidus from patients with primary polydipsia with high sensitivities and specificities >94% In hyponatremia, low copeptin levels <4 pmol/L point to primary polydipsia, high levels of
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copeptin >80 pmol/L point to hypovolemic hyponatremia In other etiologies of hyponatremia, copeptin levels overlap widely, thereby limiting its use in
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the differential diagnosis
Research Agenda:
Establish half-life time of copeptin compared to AVP Validate proposed copeptin cutoff values for the differential diagnosis of polyuria/polydipsia in large prospective studies Establish the value of copeptin measurements after hypertonic saline infusion as a new standard test in the differential diagnosis of patients with polyuria/polydipsia.
Conflict of interest statement
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MCC and WF received speaker honoraria from Thermofisher AG, the manufacturer of the copeptin assay. NGM was once employed by Thermofisher, but has currently no conflict of interest.
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38*. Katan M, Morgenthaler NG, Dixit KC, et al. Anterior and posterior pituitary function testing with simultaneous insulin tolerance test and a novel copeptin assay. The Journal of clinical endocrinology and metabolism. 2007;92(7):2640-3. 39*. Winzeler B, Zweifel C, Nigro N, et al. Postoperative copeptin concentration predicts diabetes insipidus after pituitary surgery. The Journal of clinical endocrinology and metabolism. 2015:jc20144527. 40*. Timper K, Fenske W, Kuhn F, et al. Diagnostic accuracy of copeptin in the differential diagnosis of the polyuria-polydipsia syndrome: A prospective multicenter study. The Journal of clinical endocrinology and metabolism. 2015:jc20144507. 41. Kumar S, Berl T. Sodium. Lancet. 1998;352(9123):220-8. 42. Natkunam A, Shek CC, Swaminathan R. Hyponatremia in a hospital population. Journal of medicine. 1991;22(2):83-96. 43. Upadhyay A, Jaber BL, Madias NE. Incidence and prevalence of hyponatremia. The American journal of medicine. 2006;119(7 Suppl 1):S30-5. 44. Verbalis JG, Barsony J, Sugimura Y, et al. Hyponatremia-induced osteoporosis. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 2010;25(3):554-63. 45. Chung HM, Kluge R, Schrier RW, et al. Clinical assessment of extracellular fluid volume in hyponatremia. The American journal of medicine. 1987;83(5):905-8. 46*. Fenske W, Stork S, Blechschmidt A, et al. Copeptin in the differential diagnosis of hyponatremia. The Journal of clinical endocrinology and metabolism. 2009;94(1):123-9. 47. Boursier G, Almeras M, Buthiau D, et al. CT-pro-AVP as a tool for assessment of intravascular volume depletion in severe hyponatremia. Clinical biochemistry. 2015. 48. Nigro N, Winzeler B, Suter I, et al. Copeptin in the differential diagnosis and therapy management of hyponatremia in hospitalized patients-’’The Co-MED-Study’’. Endocrine Society Meeting 2014, Chicago. 2014. 49. Nigro N, Muller B, Morgenthaler N, et al. The use of copeptin, the stable peptide of the vasopressin precursor, in the differential diagnosis of sodium imbalance in patients with acute diseases. Swiss medical weekly. 2011;141:w13270. 50. Morgenthaler N. AVP and copeptin: relationship and function, in: copeptin – Biochemistry and Clinical Diagnostics. Uni-Med Verlag. 2014:16-20. 51. Morgenthaler N. AVP and copeptin: relationship and function, in: copeptin – Biochemistry and Clinical Diagnostics. Uni-Med Verlag. 2014:28-31.
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Legends for the figures
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Figure 1 Pre Pro-Arginine Vasopressin (AVP)-Neurophysin precursor peptide, which is processed to generate a signal peptide, AVP, neurophysin II and the carboxy-terminal glycopeptide copeptin (from (50)). Figure 2 Correlation of Arginine Vasopressin (AVP) and copeptin with plasma osmolality. Plasma AVP and copeptin concentrations measured during water load and hypertonic saline tests are shown as scatter plot. rS denotes Spearman’s rank correlation coefficients (from (51)).
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Figure 3 Initial postoperative copeptin levels to predict development of diabetes insipidus after pituitary surgery. Dark grey bars represent all patients (n=205), light grey bars represent only patients where copeptin was measured within the first 12 hours postoperatively (n=157) (modified from (39)).
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Figure 4a Baseline AVP and copeptin plasma levels in the differential diagnosis of the polyuria/polydipsia Box plots depicting interquartile ranges with medians and whiskers depicting minimal and maximal values for baseline AVP (A) and copeptin (B) levels without prior thirsting in patients with complete and partial central diabetes insipidus, patients with primary polydipsia, and patients with complete and partial nephrogenic diabetes insipidus. Cut-offs for best discrimination between nephrogenic versus non-nephrogenic diabetes insipidus for AVP and copeptin are shown. The overall P value refers to Kruskal-Wallis test results across all patient subgroups. For all two-subgroup comparisons, Mann-Whitney tests were used and the P value was Bonferroni-adjusted. AVP = arginine vasopressin; DI = diabetes insipidus; PP = primary polydipsia (modified from (40)). Figure 4b Osmotically-stimulated AVP and copeptin plasma levels in the differential diagnosis of polyuria/polydipsia Box and whisker plots with medians and minimal and maximal values for stimulated AVP (A) and copeptin (B) plasma values at a plasma sodium level >147 mmol/L are depicted for patients with complete and partial central diabetes insipidus and for patients with primary polydipsia. Osmotic stimulation was provided by a combined water deprivation and saline infusion test. The cut-offs for best discrimination between primary polydipsia versus central diabetes insipidus for AVP and copeptin are depicted. The overall P value refers to Kruskal-Wallis test results across all patient subgroups. For all two-subgroup comparisons, Mann-Whitney tests were used and the P value was 16
ACCEPTED MANUSCRIPT Bonferroni-adjusted. AVP = arginine vasopressin; DI = diabetes insipidus; PP = primary polydipsia (modified from(40)). Figure 5 Proposed new algorithm for the differential diagnosis of patients with polyuria/polydipsia
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Figure 6 Copeptin in the differential diagnosis of hyponatremia. Box and whisker plots for the copeptin to U-Na+ ratio in five diagnostic groups of hyponatremic patients (n=106) and healthy controls (n=32). *, P <0.05 compared with control (ANOVA with Holm‘s post hoc test); ª, P < 0.05 compared with SIADH (ANOVA with Holm‘s post hoc test) (modified from(46)).
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