The expanding role of copeptin in the differential diagnosis of polyuria-polydipsia syndrome in children: a review

Introduction

Background

The maintenance of fluid homeostasis in humans reflects a finely tuned interplay between physiological mechanisms that stabilize plasma osmolality and effective circulating volume. The three principal determinants are: (I) water intake; (II) the thirst mechanism, which serves as the dominant, though not exclusive, regulator of fluid consumption; and (III) renal water excretion (1,2). These components operate in continuous feedback with neuroendocrine pathways to preserve internal fluid homeostasis.

At the core of this network is arginine vasopressin (AVP), also known as antidiuretic hormone (ADH), a peptide secreted by the posterior pituitary in response to changes in plasma osmolality and effective circulating volume. AVP acts primarily on the renal collecting ducts via V₂ receptors to promote water reabsorption, thereby concentrating urine and preventing excessive free-water loss. Its secretion is tightly coupled to hypothalamic osmoreceptors and cardiovascular baroreceptors, allowing integration of both plasma osmolality and effective circulating volume signals. Disruption of AVP secretion or action results in impaired urinary concentration and forms the physiological basis of the polyuria-polydipsia syndrome (PPS) (3,4).

PPS in children, characterized by hypotonic polyuria and excessive thirst, presents a distinct diagnostic challenge. The differential diagnosis includes AVP deficiency (AVP-D), primary polydipsia (PP), and, less commonly, AVP resistance (AVP-R). Accurate distinction among these entities is critical, as AVP-D in children may be associated with serious central nervous system (CNS) pathology, including inflammatory, infiltrative, or neoplastic processes (5). Misdiagnosis or delayed diagnosis may result in dehydration, electrolyte disturbances, and adverse effects on growth and neurodevelopment, while unnecessary testing may impose a significant physical and emotional burden.

Rationale and knowledge gap

Available diagnostic tests for evaluating PPS in children have limitations. The water deprivation test (WDT), traditionally considered the gold standard, is burdensome, poorly tolerated, and resource-intensive, with outcome data in children limited to small cohorts (6-9) rather than the large cohorts studied in adults (10). Its diagnostic performance is limited, particularly in distinguishing partial AVP-D from PP, due to substantial overlap in biochemical findings and urine concentrating capacity. The hypertonic saline test with AVP or copeptin measurement demonstrates superior diagnostic accuracy in adults (10), though its use in children is constrained by safety concerns and the need for intensive monitoring. Consequently, significant diagnostic uncertainty persists in routine pediatric practice.

Copeptin, the C-terminal peptide of the AVP precursor, has emerged as a practical surrogate marker of AVP secretion due to its analytical stability and reliable quantification by immunometric assays (11,12). Both random and stimulated copeptin measurements have shown promise in improving diagnostic accuracy in PPS across osmotic and non-osmotic stimulation paradigms. Recent adult studies have proposed stepwise diagnostic algorithms incorporating basal copeptin values to reduce reliance on stimulation testing (13); however, these approaches were derived exclusively from adult populations and have not been validated in children, nor do they address pediatric-specific safety and feasibility considerations.

Prior studies and reviews, including those from our group, have focused on copeptin physiology or individual stimulation tests (14-18). No pediatric-focused, laboratory-oriented synthesis currently integrates random and stimulated copeptin measurements into a coherent, stepwise diagnostic framework. Moreover, pre-analytical variability, stress-related confounding, assay-specific considerations, and inconsistent application of proposed cutoff values continue to limit clinical implementation. Addressing these gaps is essential to translate emerging copeptin data into practical, safe, and reproducible diagnostic strategies for pediatric PPS.

Objective

The objective of this review is to provide a pragmatic, pediatric-centered overview of copeptin-based diagnostics in the evaluation of PPS. We aim to synthesize current evidence on random and stimulated copeptin testing, clarify the clinical interpretation of proposed cutoff values, and present a stepwise diagnostic approach that integrates laboratory and clinical considerations relevant to ambulatory pediatric care.

AVP and copeptin: physiology and laboratory quantitation

AVP is a nonapeptide synthesized in the magnocellular neurons of the supraoptic and paraventricular nuclei of the hypothalamus. It is produced as part of a larger precursor, pre-pro-AVP, which includes neurophysin II and copeptin. During axonal transport to the posterior pituitary, pro-AVP is cleaved into its component peptides, which are stored in neurosecretory vesicles and released into the circulation in equimolar amounts in response to physiological stimuli.

The primary regulators of AVP secretion are plasma osmolality and effective circulating volume. Small increases in plasma osmolality (~1–2%) stimulate AVP release via hypothalamic osmoreceptors, promoting renal water reabsorption through aquaporin-2 insertion in the collecting ducts via V₂ receptor activation (1,3). Non-osmotic stimulation via baroreceptor pathways occurs only under conditions of marked hypovolemia (>10% volume loss) or severe hypotension and is typically inactive under normal physiological conditions (1,19). Dehydration, from any pathophysiological condition, will thus generally increase AVP/copeptin concentrations due to hyperosmolality/intracellular dehydration and less commonly as a function of hypovolemia in the event of substantial volume loss (2,19). Aside from its renal effects, AVP also acts on V₁a receptors in vascular smooth muscle, contributing to vasoconstriction (3).

Additionally, AVP is produced by parvocellular neurons in the hypothalamus, from which it is released upon stressful stimuli, to reach the anterior pituitary via the median eminence and the portal circulation. There it binds to V₁b receptors on the corticotropes, acting synergistically with CRH on these cells, to stimulate adrenocorticotropic hormone (ACTH) release (20,21).

Although direct AVP measurement by radioimmunoassay provided foundational insights into disorders of water balance, its clinical utility has been limited by preanalytical and analytical challenges, including short half-life, proteolytic degradation, loss of immunoreactivity in frozen samples, platelet-associated interference, and a labor-intensive assay methodology that achieves only moderate sensitivity (0.5–1 pg/mL) (19,22-26). These limitations have hindered widespread clinical adoption in the current clinical laboratory environment.

Copeptin, the C-terminal fragment of pre-pro-AVP, is a stable surrogate marker of AVP secretion. This 39-amino acid glycopeptide is released in equimolar amounts with AVP and correlates strongly with plasma AVP concentrations and osmolality (3,12,27-30). Unlike AVP, which is unstable and technically challenging to measure, copeptin remains stable under routine pre-analytical conditions and can be reliably quantified in unextracted serum or plasma using immunometric assays (28).

The most widely validated copeptin assay is the immunofluorimetric method performed on the B.R.A.H.M.S Kryptor platform (Thermo-Fisher Scientific, Waltham, MA, USA), which employs time-resolved amplified cryptate emission (TRACE) technology and demonstrates high analytical sensitivity (~1.1 pmol/L), broad linearity, and acceptable intra- and inter-assay variability (28,31-33). The Kryptor assay has been further validated by a good correlation with a recently developed immunoprecipitation-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) method (34). A comparable manual immunoluminometric assay [luminescent immunoassay (LIA)] yields similar results and correlates well with the Kryptor platform (32). Pediatric studies demonstrate that copeptin concentrations are generally higher in obese compared with lean children and display minimal sex-related differences until late puberty, at which point males show modestly higher concentrations than females, mirroring patterns observed in adults (16,17,28,35-37).

Alternative “research-use-only” competitive immunoassays (typically ELISA-based) have been used as lower-cost options but consistently yield substantially higher copeptin values compared with the Kryptor/Thermo-Fisher assays, reflecting limited specificity and poor diagnostic performance in PPS (32). Because these assays have not been adequately validated against the reference method, data derived from such platforms are not considered reliable for diagnostic or reference purposes. Accordingly, this review focuses exclusively on data generated using the Kryptor/B.R.A.H.M.S platform or the analogous Thermo-Fisher LIA.

The PPS

PPS is characterized by pathologically increased urine output accompanied by a proportionate rise in fluid intake. Polyuria is typically defined as >40–50 mL/kg/day in adults and, as >2 L/m²/day in children (5,38). The term “hypotonic polyuria” is often used interchangeably with PPS and emphasizes dilute urine, defined by urine osmolality (U-Osm) <300 mOsm/kg, and often well below this threshold (39). By definition, this excludes other causes of polyuria that arise from impaired concentrating capacity, such as hypokalemia, hypercalcemia, or the recovery phase of obstructive uropathy, as well as osmotic diuresis due to hyperglycemia in diabetes mellitus (2).

The differential diagnosis of PPS encompasses three major entities: AVP-D, AVP-R, and PP (38). The terms AVP-D and AVP-R are now preferred over the traditional “central/neurogenic” and “nephrogenic” diabetes insipidus (DI), respectively, as they describe the underlying vasopressin signaling defects more precisely (40). Distinguishing among these disorders, particularly differentiating partial AVP-D from PP, remains challenging. Accurate diagnosis is essential to avoid hazardous management errors (e.g., missing neuroimaging for intracranial pathology in AVP-D).

Subsequent sections review these conditions in pediatrics, referencing adult pathophysiology where relevant, followed by diagnostic approaches, including traditional water deprivation and hypertonic saline tests, and the role of copeptin measurement.

AVP-D (central or neurogenic diabetes insipidus)

AVP-D is rare in childhood and less common than PP. Etiologies include structural, infiltrative, genetic, autoimmune, and idiopathic causes (5,41-44). Destruction of AVP-producing neurons needs to be extensive (>85–90%) for AVP-D to become clinically apparent. Serum copeptin (and AVP) concentrations are very low/undetectable in complete AVP-D, they are often measurable in the early stages of AVP-D/partial AVP-D (14,15), further complicating the utility of the WDT.

Organic lesions are probably the most common and worrisome cause of AVP-D in children (45-47), most often due to CNS tumors such as craniopharyngioma, germ cell tumors or infiltrative disease such as Langerhans cell histiocytosis (LCH). PPS may be the initial presentation and precede the diagnosis of these conditions by months or years (48-50). Less frequent causes include sarcoidosis (more common in adults), tuberculous meningitis (45), pituitary stalk transection (surgical or traumatic), congenital/neurodevelopmental syndromes such as septo-optic dysplasia, holoprosencephaly, Wolfram syndrome, ROHHAD-NET (51-53).

Non-organic etiologies include AVP gene mutations (typically with autosomal dominant transmission), leading to progressive degeneration of AVP-producing neurons usually in the first decade of life (54), and autoimmune destruction of the AVP-producing neurons (i.e., neurohypophisitis) (55,56). Idiopathic AVP-D remains a diagnosis of exclusion requiring long-term follow-up, because CNS tumors or LCH may be diagnosed later (49,57-59).

AVP-R (nephrogenic diabetes insipidus)

AVP-R is a rare cause of PPS in children and is far less common than AVP-D or PP. Most pediatric cases are congenital (genetic), while acquired forms of AVP-R are predominant in adults. Serum copeptin concentrations are consistently elevated [>20 pmol/L) (60)] even in the absence of water restriction, as discussed below.

Congenital AVP-R is the most frequent and typically severe form, resulting from X-linked mutations in V₂R (61). Less commonly, AVP-R arises from Aquaporin 2 (AQP2) variants (62). Although present at birth, X-linked AVP-R is often diagnosed weeks to months after birth, especially in the absence of a positive family history (63,64). Affected male infants present with failure to thrive, irritability, and polyuria associated with large fluid (milk) intake. Severe hypernatremia (often 155–175 mEq/L) with very low U-Osm in the presence of elevated serum copeptin levels confirms the diagnosis.

Acquired AVP-R, usually seen in older children and adults, may result from several causes, including chronic lithium therapy, hypercalcemia, or hypokalemia, which impair distal tubular responsiveness to AVP. The resistance to vasopressin is typically partial and usually reversible once the underlying condition is corrected or the offending agent removed, with the possible exception of long-standing lithium exposure (4).

PP

PP is likely the most frequent cause of PPS in both children and adults. Historically termed “psychogenic polydipsia” based on early descriptions in psychiatric/behavioral disorders, contemporary cohorts show that psychiatric- or neurodevelopment-associated polydipsia accounts for only a minority of PP, prompting use of the broader, etiologically neutral term PP (10). Psychogenic and psychosis-related polydipsia remain important subtypes of PP, often characterized by a lower thirst threshold, as also noted below for dipsogenic polydipsia (65,66).

In children, the most common presentation is habitual polydipsia: excessive drinking behaviors often reinforced by parental encouragement or sweetened beverages as rewards. In adults, cultural trends promoting high fluid intake for “wellness” can similarly drive habitual or health-driven polydipsia (67). However, a percentage of subjects with PP exhibit a true defect of thirst regulation, with an abnormally low thirst threshold—dipsogenic polydipsia. This condition is also referred to as “dipsogenic diabetes insipidus (DDI)” by some investigators (66,68,69), while others use both terms interchangeably (70,71).

Serum copeptin measurements in the PPS and other conditions

The utility of circulating copeptin measurements in the differential diagnosis of the PPS has been demonstrated in several recent adult studies (11,72), redefining contemporary understanding of PPS pathophysiology and reaffirming earlier observations of AVP radioimmunoassays (19). Conversely, the diagnostic utility of copeptin measurements to diagnose inappropriate antidiuresis [syndrome of inappropriate antidiuretic hormone secretion (SIADH)] in hospitalized adults with hyponatremia has been limited (73,74), while preliminary data in children suggest moderate, yet suboptimal diagnostic sensitivity (75). Beyond water balance, copeptin has been investigated as a biomarker of endogenous stress and disease severity in a variety of conditions, including sepsis, myocardial infarction, heart failure, and stroke in adults (76,77), as well as critical illness in children (78).

Below, we review the role of serum copeptin in children with PPS under three conditions: random measurements, osmotic stimulation, and stimulation using newer non-osmotic agents.

The role of random (unstimulated) copeptin measurement in the evaluation of PPS in children

The development of the copeptin assay in 2005–2006, together with more recent advances in non-osmotic posterior pituitary stimulation tests, has expanded the diagnostic toolkit for evaluating PPS and may reduce reliance on prolonged inpatient testing in pediatric patients.

In our recent review (79), we emphasized the importance of a stepwise approach to the differential diagnosis of PPS in pediatric patients. Traditionally, the evaluation begins with measurement of serum sodium (Na), typically combined with plasma osmolality and U-Osm under unrestricted fluid intake or after a brief period of water restriction. Despite some variability of serum sodium and copeptin concentrations related to the hydration status (36,37), hypernatremia (serum Na ≥147 mEq/L) accompanied by inappropriately low U-Osm (<300 mOsm/kg) strongly supports a diagnosis of AVP-D or AVP-R, which may be distinguished by copeptin measurement. However, because most patients maintain an intact thirst mechanism and can drink to maintain normonatremia, increased Na concentrations are uncommon in random samples, and this approach is often non-diagnostic.

An important question is whether a random copeptin concentration (in the absence of hypernatremia) can obviate the need for further dynamic testing. At our center, we utilize random measurements strictly as a screening tool. At the lower end of the spectrum, random copeptin has limited diagnostic accuracy. While undetectable or very low concentrations (≤2.3–3.5 pmol/L) are characteristic of complete or severe AVP-D (37), these values can also overlap with findings in healthy children or those with PP (15,18). Conversely, random copeptin levels may serve as a useful screening tool at the higher threshold levels meant to identify or exclude AVP-D. To avoid the devastating risk of missing an organic pituitary lesion causing partial AVP-D, the screening cutoff must prioritize high sensitivity over specificity. Although previous studies have reported random copeptin values not exceeding 5.0 pmol/L in partial AVP-D, this threshold is derived from limited cohorts of affected children (14,15,61). Moreover, considerations must be made for some variability in measured concentrations between individuals, due to differences between the Kryptor automated assay and the LIA, as well as the inter-assay coefficients of variation (CV) (32). To account for these variables, our center employs a conservative, albeit arbitrary, cutoff of 6.0 pmol/L, below which we consider AVP-D to be unlikely. Consequently, our diagnostic algorithm recommends routine dynamic testing for any value below this threshold, whereas dynamic testing for values at or above 6.0 pmol/L is performed selectively, if considered clinically indicated by the provider’s assessment of the patient’s presentation.

Conversely, random copeptin values ≥20 pmol/L have been considered diagnostic of AVP-R in adults (10) and children (60) with PPS. However, we found that ~5–10% of short, otherwise healthy, children without PPS have very elevated serum copeptin concentrations in random samples, most likely due to the pain or stress associated with venipuncture. These values decline with time, but may remain elevated for at least the first hour (80); therefore, waiting 20–30 min to repeat sampling, while often lowering copeptin concentrations (81), will not “normalize” them if they were very elevated initially. As such, random copeptin levels ≥20 pmol/L in children should always be interpreted in conjunction with clinical and laboratory findings and confirmed through subsequent testing when indicated.

The clinical interpretation of random copeptin thresholds and their intended use as screening, reassurance, or diagnostic indicators are summarized in Table 1.

Traditional dynamic tests for evaluation of PPS in children (based on osmotic stimulation of AVP/copeptin)

When random laboratory results are inconclusive, the next step involves a dynamic test. Until recently, the available osmotic stimulation tests included the WDT, the current gold standard for PPS evaluation in children, and the hypertonic saline infusion test (HST or HSIT).

The WDT

The WDT is based on the physiologic rise in AVP secretion during water restriction in AVP-sufficient children, which leads to reduced urine output and increased U-Osm above a defined threshold, usually set at 800 mOsm/kg for children ≥2 years (and somewhat lower threshold in younger children) with maintenance of normal serum Na and plasma osmolality (15). In AVP-D, hypotonic polyuria (U-Osm <300 mOsm/kg) persists, resulting in rising serum sodium and eventually leading to hypernatremic dehydration. The WDT has noticeable limitations. First, the WDT requires an inpatient admission for close monitoring due to the prolonged fluid restriction (typically 8–12 hours), particularly in children <4–5 years who are at higher risk for dehydration and hypoglycemia. The water restriction is often both burdensome and truly distressing for the (thirsty) child and their accompanying caregiver. Second, the WDT has limited diagnostic accuracy, reported at ~76% in adults (10) and only 53% in a recent pediatric cohort (47), with only modest improvement using optimized cutoffs for U-Osm in the latter study. Performance is particularly poor in differentiating partial AVP-D from PP (15,60), as residual AVP secretion and reduced glomerular filtration rate during dehydration may allow patients with partial AVP-D to decrease their urinary output and achieve intermediate U-Osm values that overlap with those observed in PP (60,71,82,83). End-of-test serum copeptin measurements also show substantial overlap and do not improve diagnostic yield in either adults or children (10,14,60). In practice, the WDT is most useful when a complete AVP-D or AVP-R is strongly suspected from the history (i.e., abrupt onset, nocturnal drinking/urination), in which case just a few hours of water restriction generally corroborate the diagnosis, as suggested decades ago with the use of traditional osmolality parameters (7). In this category of patients, we have consistently observed that water-restriction for a short 3–6-hour interval raises serum sodium ≥147 mmol/L, which, in the face of persistently hypotonic urine, supports diabetes insipidus. Serum copeptin quantitation will then help distinguish AVP-D from AVP-R.

The HST or HSIT

The HST, using a 3% NaCl solution, is a physiologically based test of posterior pituitary stimulation. Unlike the WDT, it induces hypernatremia and plasma hyperosmolality rapidly, typically within a few hours, without causing plasma volume contraction (indeed, it produces mild expansion) (19). This results in robust stimulation of AVP and copeptin secretion, without effects on urine output related to volume depletion. The HST is considered the gold standard test for evaluation of PPS in adults (10,84).

Although diagnostically valuable in children, the test has been used infrequently in the pediatric population (44,85,86). Drawbacks include concerns about the potential effects of a rapid rise in plasma osmolality on the developing brain and the requirement for frequent serum sodium monitoring (every 30 min), which would demand intensive-care-level supervision. Additionally, children (like adults) experience pronounced thirst and often general malaise during the infusion. A recent study of 27 children suggested high efficacy of the HST, although unclear methodological details limit interpretation (87). These limitations and concerns have prevented HST from being widely adopted as a standard diagnostic test in children.

The new dynamic tests for evaluation of PPS in children (based on non-osmotic stimulation of AVP/copeptin)

Historically, the assessment of posterior pituitary function has relied on the osmotic stimulation of AVP/copeptin via the WDT or the HST. While these remain the traditional standards, recent research has explored newer diagnostic approaches using non-osmotic agents to stimulate copeptin.

These pharmacological agents include insulin, arginine hydrochloride, glucagon, and levodopa. Notably, while these agents are established growth hormone (GH) secretagogues, their mechanisms for copeptin stimulation remain incompletely understood—unlike the well-defined physiological pathways of osmotic stimulation (15,16,88). Interestingly, this stimulatory effect is not universal among GH secretagogues; both clonidine and macimorelin appear to have no impact on copeptin secretion, highlighting a distinct divergence in their neuroendocrine pathways (89-91).

Copeptin stimulation tests by single (non-osmotic) agent

The arginine stimulation test

Intravenous arginine hydrochloride (0.5 g/kg over 30 min) has demonstrated diagnostic accuracy of approximately 75% in adults with PPS—comparable to the WDT and lower than that reported for the HST (~95%) (84). Its copeptin-stimulating potency is lower in children than in adults, as demonstrated in the original report (92), and confirmed in subsequent reports, which showed peak copeptin responses ranging from 7 to 12.2 pmol/L (14,17,18), with inter-study variability.

Proposed arginine-stimulated copeptin cutoffs for differentiating AVP-D from PP in children generally fall within the 3.0–3.8 pmol/L range (14,17,18), overlapping with thresholds proposed for random copeptin measurements (37). Although median peak copeptin concentrations are typically higher in PP than in AVP-D, substantial overlap between individual values limits diagnostic discrimination. Moreover, most pediatric cohorts have been small, with relatively few participants with AVP-D and a predominance of complete rather than partial AVP-D cases (79). Taken together, available data suggest that arginine stimulation alone may have limited ability to reliably distinguish partial AVP-D from PP in children.

The insulin tolerance test (ITT)

Insulin-induced hypoglycemia to a serum glucose concentration <40 mg/dL (2.2 mmol/L) has been known to stimulate AVP secretion (93). Likewise, it was shown to stimulate copeptin in a study of adults with prior pituitary surgery or pituitary disease, in which a “control” group with intact posterior pituitary function exhibited an approximately 200% increase in copeptin levels over baseline, with a peak of 11.2 pmol/L, whereas patients with established AVP-D showed a blunted response (94). Considerable interindividual variability was noted among controls, several of whom displayed only minimal copeptin elevations. In children, insulin-induced hypoglycemia has been associated with a more modest copeptin increase of approximately 80–110% above baseline, with median peak concentrations reaching up to 8.4 pmol/L, again accompanied by substantial inter-subject variability and overlapping responses between diagnostic groups (88,95). Due to the variability in copeptin responses and the procedural risks associated with inducing hypoglycemia, the ITT has been used infrequently as a posterior pituitary stimulation test in children.

The glucagon stimulation test

Glucagon, when given intramuscularly or subcutaneously [but not intravenously (96)], has shown promising results in adult cohorts as a non-osmotic stimulus of copeptin secretion. It appears to elicit a more robust copeptin peak concentration (median 11.5–12.1 pmol/L, approximately +175%) than arginine IV, achieving good diagnostic accuracy in the differential diagnosis of PPS (90,97). In another adult cohort of 32 healthy subjects, the post-glucagon copeptin peak was more than twice as high (98). The discrepancy in peak copeptin values between these studies may reflect differences in study design, subject selection, or the use, in the studies with lower responders, of antiemetic medications, which could potentially blunt the copeptin response (99,100). Only recently the first pediatric evaluation of glucagon-stimulated copeptin, published as part of a non–peer-reviewed doctoral thesis (101), showed that IM glucagon elicited a mean peak copeptin concentration of 10.6 pmol/L—comparable to the values observed in the adult studies by Atila et al. (90,97) In 20 short-statured children without PPS, with a trend toward lower responses in the small subgroup (N=4) diagnosed with GH deficiency. Thus, glucagon effectively stimulates copeptin secretion, though pediatric data remain limited.

The levodopa (L-Dopa) stimulation test

Levodopa, another GH-stimulating agent, has recently been evaluated as a potential stimulus for copeptin secretion in a cohort of control children with short stature. In this study, the median peak copeptin concentration reached 19.4 pmol/L, however approximately 10% of participants showed no measurable response (102). In the subset of children who underwent both the levodopa test and the ITT, the median copeptin peak following levodopa administration (34.6 pmol/L) was significantly higher than that induced by hypoglycemia (8.9 pmol/L).

Although levodopa was not directly compared with arginine as a copeptin-stimulating agent, the copeptin peaks observed in this pediatric cohort exceeded those reported for arginine in prior studies (14,17,18). Nevertheless, response variability, including absent responses in a subset of participants, and the lack of data in children with PPS underscore the need for further validation before levodopa-based testing can be considered for diagnostic application in PPS.

Copeptin stimulation tests by dual (non-osmotically active) agents

During our investigations of copeptin secretion in children, we examined whether combining two non-osmotic copeptin-stimulating agents could augment copeptin release beyond that observed with single-agent stimulation. To explore this question, in our previous studies, we utilized (as “controls”) cohorts of short, otherwise healthy children without symptoms of PPS undergoing GH stimulation testing with arginine combined with either insulin [arginine-insulin tolerance test (AITT)] or levodopa/carbidopa [arginine-levodopa/carbidopa stimulation test (ALD-ST)].

The AITT

In this standard test of GH stimulation, intravenous arginine hydrochloride (0.5 g/kg administered from 0–30 min) is followed by an intravenous bolus of regular insulin (0.1 IU/kg) at 60 min, with serial blood sampling through 120 min (103). In our cohort, serum copeptin increased modestly during the first 60 min following arginine infusion, followed by a consistent peak (≥10 pmol/L in 97% of subjects) between 80 and 90 min, corresponding temporally to hypoglycemia (16). Optimal sampling times were identified at 0, 80, and 90 min. The magnitude of the peak response (median 23 pmol/L at 90 min) exceeded values historically reported for arginine or insulin alone, consistent with at least an additive effect of combined stimulation (Figure 1).

Figure 1 Copeptin responses to dual-agent stimulation across diagnostic groups. Data are presented as median (interquartile range) for both baseline and peak copeptin concentrations (pmol/L). Symbols represent individual participant data points beyond the whisker limits (1.5x interquartile range). AITT, arginine-insulin tolerance test; ALD-ST, arginine-levodopa-stimulation test; AVP-D, arginine-vasopressin deficiency; PP, primary polydipsia.

Although the AITT demonstrated reproducible copeptin peak timing, it has not been validated for the differential diagnosis of PPS in children. Unfortunately, procedural complexity, monitoring requirements, and hypoglycemia risk limit its adoption for routine pediatric diagnostic use.

The ALD-ST

Our group has examined copeptin dynamics during the ALD-ST in cohorts of 47 healthy short children as controls and in 20 children with PPS (15). We modified this standard GH secretion test in children (104,105) to allow the use of the levodopa/carbidopa combination (106), as an individual levodopa preparation is not commercially available in the U.S. In this test, levodopa/carbidopa is given orally (150–200 mg/m²), simultaneously with the beginning of a 30-min period. infusion of 10% arginine hydrochloride (0.5 g/kg), followed by serum copeptin measurements at 0, 60, 90, and 120 min (15). Although the timing of the peak copeptin response was more variable and less predictable than in the AITT, possibly due to variable bioavailability of oral levodopa (107), the overall peak response was similarly robust (Figure 1).

Copeptin responses were significantly higher in approximately 50% of patients who experienced nausea or vomiting during testing compared with those who did not (median peak 131 vs. 22.7 pmol/L; P=0.001). In the combined cohort, the median peak was 44 pmol/L, and all controls achieved peak values ≥10 pmol/L. Children with complete AVP-D showed minimal responses (<3 pmol/L), whereas those with partial AVP-D demonstrated a wide range of responses, including values overlapping with those observed in non-deficient participants. Within this cohort, a stimulated copeptin cutoff of 9.3 pmol/L demonstrated optimal discriminatory performance for differentiating AVP-D from PP (15).

The findings observed in these studies of non-osmotic stimulation suggest that combining two agents produces consistently stronger copeptin responses than single-agent stimulation. The aforementioned study from a single center suggests favorable diagnostic performance and tolerability of ALD-ST, though prospective multicenter validation will be required before broader clinical adoption.

An important limitation of non-osmotic stimulation testing, including the ALD-ST, is the influence of nausea and vomiting on copeptin secretion. Nausea represents a potent non-osmotic stimulus for AVP/copeptin release and may exaggerate copeptin responses, potentially reducing diagnostic specificity in partial AVP-D. Accordingly, interpretation of results from the ALD-ST, the HST (108), and potentially other copeptin stimulation tests should incorporate careful documentation of test-related symptoms. Future studies should assess symptom-stratified cutoff values to optimize diagnostic performance.

The distribution of copeptin responses across diagnostic groups during the dual-agent stimulation tests is illustrated in Figure 1, highlighting the robust responses observed in controls and PP and the markedly blunted responses characteristic of AVP-D. A comparative summary of osmotic and non-osmotic copeptin stimulation tests, including their advantages, limitations, pediatric feasibility, and proposed diagnostic cutoffs, is provided in Table 2.

The role of magnetic resonance imaging (MRI) of the pituitary and hypothalamus in the investigation of PPS in children

MRI has major diagnostic relevance in complementing laboratory investigations for the evaluation of PPS in children. In healthy children and adults, the posterior pituitary appears as a hyperintense signal, referred to as the posterior pituitary hyperintense signal (PPHS) or “bright spot”, on T1-weighted MRI images. The absence of PPHS has been shown to correlate with AVP-D (109). It has been suggested that MRI could be used as a first-line screening tool for PPS in adults (110). However, a review of a large adult cohort demonstrated that MRI has only moderate diagnostic accuracy (~75%) for distinguishing PPS subtypes in this age group (10).

Pediatric data are more limited but suggest potentially higher diagnostic sensitivity and specificity in children, with reported diagnostic accuracy approaching 95% in selected cohorts (111). Despite its utility, MRI is costly, not readily available in all settings, and often requires sedation or general anesthesia in younger children. For these reasons, MRI is not recommended as a first-line test in the initial evaluation of pediatric PPS. Nevertheless, it remains an indispensable tool in several situations such as (I) children with symptoms suggestive of organic intracranial pathology (i.e., tumors, malformations, infiltrative or vascular lesions); (II) neonates, infants and children biochemically diagnosed with AVP-D, to define etiology and exclude structural abnormalities; and (III) any child with an uncertain diagnosis after biochemical and dynamic testing.

A sequential, graded approach to the child with PPS

Figure 2 illustrates a stepwise diagnostic approach used in our division for children presenting with PPS. Once hypotonic polyuria and concomitant polydipsia are confirmed, initial evaluation includes screening laboratory tests obtained under fasting conditions, ensuring, however, that fluid deprivation does not exceed the duration the child typically tolerates at home (which could be as little as 2 hours).

Figure 2 Recommended steps in the diagnostic evaluation of PPS in children with normal serum sodium concentrations/normal plasma osmolality. This figure originates from Garibaldi et al. (79), and is reproduced/adapted under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) license. ALD-ST, arginine-levodopa/carbidopa stimulation test; AVP-D, arginine-vasopressin deficiency; AVP-R, arginine vasopressin resistance; MRI, magnetic resonance imaging; PPS, polyuria-polydipsia syndrome; WDT, water deprivation test.

AVP-R should be considered early in the evaluation of children with hypotonic polyuria. Markedly elevated random copeptin concentrations are suggestive of AVP-R and, with the caveat of spurious copeptin elevations observed in a minority of children (as discussed above), support the diagnosis without copeptin stimulation testing. Incorporating AVP-R into the initial diagnostic framework can help avoid further, unnecessary diagnostic procedures and facilitate timely evaluation of underlying renal or genetic etiologies.

If initial testing is inconclusive, as often occurs in children with preserved thirst mechanisms, an outpatient ALD-ST may be performed. In our single-center pediatric study, this test has demonstrated promising diagnostic performance. In our cohort, a peak copeptin response ≥9.3 pmol/L showed optimal discriminatory performance for differentiating AVP-D from PP (15). However, because peak values ≥10 pmol/L were observed in a small number of children with complete or partial AVP-D, and approximately 94% of controls exhibited peak responses ≥15 pmol/L, a threshold of ≥15 pmol/L may provide greater reassurance against both complete and partial AVP-D. This conservative (albeit arbitrary) cutoff prioritizes diagnostic sensitivity over specificity with regard to excluding a diagnosis of AVP-D, and its potentially serious consequences in the presence of organic CNS pathology. Key copeptin thresholds used within this stepwise diagnostic approach, and their recommended clinical interpretation, are summarized in Table 1.

When copeptin responses are suboptimal or equivocal, or when neurological concerns are present, brain MRI (with both non-contrast and contrast images) should be pursued to assess the PPHS, structural hypothalamic-pituitary abnormalities, thickening of the pituitary stalk, neoplasms, etc.

At this point of the diagnostic evaluation, the WDT often adds limited incremental diagnostic value. Nevertheless, as the WDT remains widely regarded as the pediatric gold standard in many institutions, it may still be used selectively. Specifically, it may serve as an abbreviated first-line test when the clinical history strongly suggests severe AVP-D, or as a second-line assessment in children with suspected partial AVP-D to evaluate tolerance to fluid restriction. This information can be useful for family counseling, particularly during intercurrent illnesses associated with reduced intake or increased fluid losses. This stepwise, physiologically based approach has been associated with a high diagnostic yield while decreasing the percentage of WDTs (and related hospitalizations) in our single-center experience.

Limitations and future directions

Despite growing interest in copeptin-based diagnostics for PPS, several important limitations should be acknowledged. First, much of the pediatric evidence supporting random and stimulated copeptin measurements originates from single-center studies with relatively small sample sizes. While these studies provide valuable mechanistic and proof-of-concept data, their findings may not be fully generalizable across diverse pediatric populations, clinical settings, and laboratory environments.

Second, pediatric-specific diagnostic thresholds for copeptin remain incompletely standardized. Reported cutoff values vary depending on study design, stimulation paradigm, assay platform, and sampling conditions. Pre-analytical factors, including hydration status, acute stress, and nausea, may further influence copeptin concentrations and complicate interpretation, particularly for random measurements.

Third, direct comparative data between different stimulation tests in children are limited, and most diagnostic algorithms rely on extrapolation from adult studies or indirect comparisons across pediatric cohorts. As a result, the proposed stepwise diagnostic approach should be viewed as a pragmatic framework rather than a definitive guideline.

Future research should prioritize prospective, multicenter pediatric studies to validate copeptin thresholds, compare stimulation paradigms head-to-head, and assess real-world diagnostic performance across diverse patient populations. While standardization of assay methodology is currently assured using one of the Thermo-Fisher immunometric assays, standardization of sampling protocols will be essential to facilitate broader clinical implementation.

Concluding remarks

The increasing availability of copeptin assays, together with the development of novel stimulation protocols, has expanded the diagnostic options for evaluating children with PPS. Copeptin-based testing offers practical advantages over direct AVP measurement and addresses some of the limitations associated with the traditional WDT.

Among available non-osmotic dynamic tests, the outpatient ALD-ST has demonstrated good accuracy for PPS diagnosis in our single-center study. However, as no single test is sufficient to fully characterize all etiologies of PPS, we propose a sequential, integrative diagnostic approach in which multiple complementary assessments, including copeptin-based testing, are applied in a stepwise manner.

Acknowledgments

None.

Footnote

Provenance and Peer Review: This article was commissioned by the Guest Editor (Kay Weng Choy) for the series “Copeptin Testing: Current Applications and Emerging Insights” published in Journal of Laboratory and Precision Medicine. The article has undergone external peer review.

Peer Review File: Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-2025-1-77/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-2025-1-77/coif). The series “Copeptin Testing: Current Applications and Emerging Insights” was commissioned by the editorial office without any funding or sponsorship. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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