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COPEPTIN, A STABLE PEPTIDE OF THE ARGININE VASOPRESSIN PRECURSOR, IS ELEVATED IN HEMORRHAGIC AND SEPTIC SHOCK

Morgenthaler, Nils G.*; Müller, Beat; Struck, Joachim*; Bergmann, Andreas*; Redl, Heinz; Christ-Crain, Mirjam

doi: 10.1097/SHK.0b013e318033e5da
Basic Science Aspects
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Arginine vasopressin (AVP) levels are increased in hemorrhagic and septic shock. Measurement of AVP levels has limitations due to its short half-life and cumbersome detection method. Copeptin is a more stable peptide derived from the same precursor molecule. We evaluated the plasma copeptin concentration in two independent studies: first, in an experimental baboon model of hemorrhagic shock, and second, in a prospective observational study of 101 consecutive critically ill patients at a university hospital. Copeptin was measured with a newly developed sandwich immunoassay using two polyclonal antibodies to the C-terminal region (amino acid sequence 132-164) of pre-pro-AVP. Copeptin concentrations in hemorrhagic shock increased markedly from median (range) of 7.5 [2.7-13) to 269 pM (241-456 pM). After reperfusion, copeptin levels dropped within hours to a plateau of 27 pM (15-78 pM). In the critically ill patient cohort, copeptin values increased significantly with the severity of the disease and were in patients without sepsis [27.6 pM [2.3-297 pM]), in sepsis [50.0 pM [8.5-268 pM]), in severe sepsis [73.6 pM [15.3-317 pM]), and in septic shock [171.5 pM (35.1-504 pM] compared with 4.1 pM (1.0-13.8 pM) in healthy controls (P for all vs. controls <0.001). On admission, circulating copeptin levels were higher in nonsurvivors (171.5 pM, 46.5-504.0 pM) as compared with survivors (86.8 pM, 8.5-386.0 pM; P = 0.01). Copeptin levels correlated with basal cortisol levels (r = 0.42; P < 0.001) and osmolality (r = 0.42; P < 0.001). In a logistic regression model including other covariates besides copeptin (e.g., determinants of fluid status) on survival, serum copeptin levels were the only independent significant predictor of outcome (P = 0.03). Copeptin concentrations are elevated in hemorrhagic and septic shock. Copeptin was higher on admission in nonsurvivors as compared with survivors, suggesting copeptin as a prognostic marker in sepsis. The availability of a reliable assay for the measurement of AVP release can also prove useful for the assessment of fluid and osmosis status in various diseases.

*Research Department, B R A H M S AG, Biotechnology Centre Hennigsdorf/Berlin, Germany; Division of Endocrinology, Diabetology and Clinical Nutrition, Department of Internal Medicine, University Hospital Basel, Basel, Switzerland; and Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria

Received 5 Sep 2006; first review completed 26 Sep 2006; accepted in final form 8 Dec 2006

Address reprint requests to Nils G. Morgenthaler, Research Department, B R A H M S AG, Neuendorfstraβe 25, D-16761 Hennigsdorf bei Berlin, Germany. E-mail: n.morgenthaler@brahms.de.

The authors Morgenthaler and Müller, contributed equally to the study.

N.G.M., J.S., and AB are employees of B R A H M S AG, a company presently developing the copeptin assay.

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INTRODUCTION

Arginine vasopressin (AVP), also known as the antidiuretic hormone (ADH), is a nonapeptide with a plethora of functions, including endocrine, hemodynamic, and osmoregulatory effects (1-3). It is produced in the hypothalamus, transported to the posterior lobe of the pituitary gland, and secreted upon hemodynamic and osmotic stimuli. Arginine vasopressin exerts its peripheral effects through three different types of receptors: V1a, V1b, and V2. The disturbance of AVP release contributes to the pathogenesis of several diseases, among them diabetes insipidus and, in case of excess AVP, the syndrome of inappropriate ADH secretion (2, 4).

Secondary involvement of AVP in the pathogenesis of other diseases such as congestive heart failure has repeatedly been reported (5). Arginine vasopressin is also considered part of the endocrine stress response to cardiac arrest and shock (6), and the therapeutic role of AVP administration is presently the focus of experimental and clinical trials in cardiac arrest and different shock states (7, 8).

In this context, improved knowledge of endogenous plasma AVP levels would be helpful to diagnose pathologies of the osmotic system and guide or control treatment in patients with cardiovascular diseases. However, the methodological reliability of laboratory assays determining plasma AVP concentrations is problematic. More than 90% of AVP in the circulation is bound to platelets (9), resulting in either underestimation or, in case of prolonged storage of unprocessed blood samples, in falsely elevated or varying AVP levels (9, 10). Furthermore, AVP is unstable in isolated plasma even when stored at −20°C (11), and due to its small size, AVP cannot be measured by sandwich immunoassays but needs competitive assays, which are generally time-consuming. Accordingly, AVP results usually are available several days after blood withdrawal and are principally inferior regarding analytical sensitivity. Thus, despite its pivotal physiological role, routine measurement of circulating AVP levels can never be implemented in clinical and acute patient care, but has been reserved for specific endocrine questions where the use has been well described over the last decades (12, 13).

Arginine vasopressin derives from a 166-amino acid-long precursor protein, pre-pro-vasopressin, which consists of a signal peptide, AVP, neurophysin II, and copeptin (14). Copeptin, the C-terminal part of the AVP precursor, is a 39-amino acid-long glycosylated peptide with a leucine-rich core segment (15). It is produced together with AVP in an equimolar ratio and processed on the way from the hypothalamus to the pituitary (16), during which it may act together with neurophysin II as a carrier protein of AVP. Recent data suggest an important role of copeptin in the correct structural formation of the AVP precursor as a prerequisite for its efficient proteolytic maturation (17).

The establishment of a reliable detection method to assess copeptin levels in the circulation offers the possibility to use this peptide as a surrogate marker for mature AVP (18). Because of its stoichiometric synthesis, copeptin should represent the release of AVP, a situation quite comparable to that of insulin and C-peptide. Following this, we developed a sandwich immunoassay for the measurement of copeptin in plasma and serum (19). Arginine vasopressin and copeptin showed a very good correlation in plasma samples of healthy individuals and patients (19). The finding that copeptin is stable ex vivo for days at room temperature simplified the measurement and helped to overcome the shortfalls of AVP detection.

The purpose of this study was to examine patterns of copeptin release in two independent settings of shock. In both situations, elevations of AVP have been described before. As a first setting, we took an experimental baboon model of hemorrhagic shock, and in a second independent study, we measured copeptin in a cohort of critically ill patients with increasing severity of septic shock. We aimed to evaluate if copeptin followed the release pattern described for mature AVP in these conditions (20-23).

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MATERIALS AND METHODS

Baboon model of hemorrhagic shock

The present retrospective study was performed in adult male baboons (Papio ursinus) weighing between 23 and 33 kg. The animals were caged at the nonhuman primate unit of the Biocon Research LTD Laboratory in Pretoria, South Africa, for at least 3 months. The clinical condition of the baboons had been checked before their entry into quarantine, and at the beginning of the study, all the animals were free of disease.

The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Biocon Research Laboratories. The animals were treated in accordance with National Institutes of Health guidelines.

Before the experiment, animals were fasted overnight. All the animals were anesthetized and placed on a ventilator. Throughout the experiment, the baboons were ventilated with continuous positive airway pressure of 5 cm water to keep the alveoli open. Continuous end-tidal carbon dioxide measurements were used to monitor ventilation, and the inspired oxygen content (FIO2) was 0.25 ± 0.02. Subsequent anesthesia was maintained with i.v. pentobarbital (2-5 mg kg−1 h−1). After adequate anesthesia was obtained, the following catheters were placed; (1) an arterial line for monitoring blood pressure and for obtaining blood samples, (2) a femoral vein catheter for blood withdrawal, and (3) a urinary catheter for measuring urine production. Temperature was maintained between 36.8°C and 37.5°C.

After instrumentation was completed, the animals were allowed to stabilize for 20 min. The animals were then bled down to a MAP of 40 mmHg in two stages. Over the first 30 min, the MAP was reduced to 60 mmHg. The MAP was then reduced from 60 to 40 mmHg over the next 30 min. The MAP was kept at 40 mmHg until the base excess reached −5 or the shock period reached 3 h. Base excess was used as the criteria for ending the shock period and initiating volume resuscitation because this is a physiologically relevant marker of the magnitude of the shock state. During the first hour of resuscitation, the MAP was brought to 100 mmHg by the infusion of 25% of the shed blood plus Ringer solution, and during the third hour of resuscitation, an additional 25% of the shed blood plus Ringer solution was administered to raise the MAP to baseline levels. Blood samples were collected before hemorrhage 1 and 2 h after the onset of hemorrhage, at the end of hemorrhage (onset of resuscitation = R 0), and at 1, 2, 4, and 9 h after the onset of resuscitation (R1, R2, R4, R9). Serum and plasma samples were prepared and aliquots frozen at −80°C for further analysis.

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Patients

Intensive care unit patients

We evaluated copeptin concentrations in 101 consecutive critically ill patients admitted to the medical intensive care unit (ICU) of the University Hospital of Basel, Switzerland. The primary objective of this study was the assessment of the prognostic value of endocrine dysfunctions in critically ill patients. Details of the characteristics of the study population and study design and definitions used have been reported in detail elsewhere (24-26). Briefly, over a 9-month period, 101 consecutive patients, including neutropenic and immunosuppressed patients, admitted to the medical ICU were included. Patients were followed until hospital discharge or death. For the purpose of this study, in-hospital mortality was used as end point. Vital signs, clinical status, and severity of disease as well as all laboratory parameters, including basal cortisol and copeptin levels, were assessed on admission, and commonly used physiological scores were calculated (acute physiology and chronic health evaluation [APACHE II] score and Simplified Acute Physiology Score [SAPS II]). When feasible, consent was obtained before enrollment in conscious patients; otherwise, the consent was obtained from the patients' next of kin. The study protocol had prior approval by the hospital's ethical review board.

Patients were classified at the time of blood collection into systemic inflammatory response syndrome [SIRS], sepsis, severe sepsis, and septic shock, defined according to well-known consensus criteria (27). Systemic inflammatory response syndrome was characterized by the presence of at least two of the following four clinical criteria: (1) fever or hypothermia (temperature >38°C or <36°C), (2) tachycardia (>90 beats per minute), (3) tachypnea (>20 breaths per minute or <32 mmHg or the need for mechanical ventilation support); (4) an altered white blood cell count of more than 12,000 cells per microliter, less than 4,000 cells per microliter, or the presence of more than 10% band forms, respectively. Sepsis was defined as SIRS with an infection. Infection was diagnosed according to standardized criteria or, in case of uncertainty, by an infectious disease specialist. This ascertainment was done retrospectively on the basis of the review of the complete patient charts, results of microbiological cultures, chest radiographs, and, when available, postmortem examination reports. Severe sepsis was defined as the presence of sepsis and at least one of the following manifestations of organ failure: (1) hypoxemia (PaO2 of <75 mmHg), (2) metabolic acidosis (pH of <7.30), (3) oliguria (output <30 mL h−1), (4) lactic acidosis (serum lactate level of >2 mM), and (5) an acute alteration in mental status without sedation (reduction by >3 points from baseline value in the Glasgow Coma score). Septic shock was defined as the presence of sepsis accompanied by a sustained decrease in systolic blood pressure (<90 mmHg or a drop of 40 mmHg from baseline systolic blood pressure) despite fluid resuscitation and the need for vasoactive amines to maintain adequate blood pressure. An isolated microorganism was considered to be pathogenic if recovered within a 24-h period before or after the onset of the systemic response. Colonizations with bacteria (e.g., asymptomatic bacteriuria in a patient with bladder catheter without leukocyturia) or postmortem positive blood cultures were disregarded. Microbiological tests and antibiotic therapy were prescribed by physicians on duty according to the usual practice without interference by the research team.

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Healthy controls

For comparative purposes, copeptin values were also measured in 84 age- and sex-matched healthy blood donors with no history or clinical evidence of acute or chronic disease.

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Assays

Blood was obtained from an indwelling arterial or venous catheter. Results of the routine blood analyses (i.e., complete blood count and serum chemistry, including C-reactive protein [CRP], blood gas analyses, and osmolality) were recorded. The blood was separated into plasma at the time of blood draw and frozen at −70°C until assay. Measurement was done in a blinded fashion as a batch analysis.

Serum copeptin levels were measured with a new sandwich immunoassay (B R A H M S AG, Hennigsdorf/Berlin, Germany] as described in detail previously (19). Briefly, this sandwich immunoluminometric assay uses two polyclonal antibodies to the C-terminal region (amino acid sequence 132-164) of pre-pro-AVP. One antibody is bound to polystyrene tubes, and the other is labeled with acridinium ester for chemiluminescence detection. The assay requires 50 μL of either serum or plasma; no extraction steps or other preanalytical procedures such as addition of protease inhibitors are necessary, and results are available in approximately 3 h. The analytical detection limit is 1.7 pM, and the total precision of the assay (interlaboratory coefficient of variation [CV]) was less than 20% for copeptin concentrations across the calibration curve (up to 405 pM). Copeptin plasma concentration in 359 healthy individuals had a median of 4.2 pM (range, 1-13.8 pM). The 97.5th percentile was 11.25 pM, and the 2.5th percentile was 1.7 pM. Of all 359 tested volunteers, only 9 (2.5%) had a plasma copeptin concentration less than the detection limit of the assay of 1.7 pM. Importantly, in contrast to mature AVP, copeptin is very 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 7 days at room temperature and 14 days at 4°C (19).

C-Reactive protein was determined by a routinely used enzyme immunoassay (EMIT; Merck Diagnostica, Zurich, Switzerland, reference range <10 mg L−1; intra-assay CV, 0.9%/1.4%/11.4%; and interassay CV, 2%/2%/26.3% at CRP levels of 33.6/57.6/150 mg L−1, respectively). Serum interleukin (IL) 6 concentrations were measured with a commercially available quantitative sandwich enzyme immunoassay (CLB, Pelikine Compact, Amsterdam, Netherlands, reference range, <3.12 pg mL−1; intra-assay CV, 4.2%/1.6%/2.0%; and interassay CV, 6.2%/3.3%/3.8% at IL-6 levels of 16.8/97.7/186 pg mL−1, respectively). Procalcitonin levels were assessed with an immunoluminometric assay (LUMItest PCT; B R A H M S AG; lower detection limit, 0.1 μg L−1, intra-assay CV, 6.3% and 2.7% at ProCT levels of 0.4 and 43.2 μg L−1; interassay CV, 13.4% and 7.1% at ProCT levels of 0.5 and 34.2 μg L−1). The functional detection limit of this assay is 0.3 μg L−1).

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Statistical analysis

Data are given as mean ± standard deviation (SD) or median (range), respectively. Frequency comparison was done by chi-square test. Two-group comparison was performed nonparametrically by the Mann-Whitney U test. For multigroup comparisons, Kruskal-Wallis one-way ANOVA was used with least square difference posthoc evaluation. Receiver-operating characteristics (ROC) were calculated using MedCalc for Windows [version 7.2.1.0; Mariakerke, Belgium). Levels that were nondetectable were assigned a value equal to the lower limit of detection for the assay. All statistical tests were two-tailed, and P values less than 0.05 were considered to indicate statistical significance. Correlation analyses were performed by using Spearman rank correlation.

To assess the influence of copeptin and other covariates (e.g., determinants of fluid status) on survival, a logistic regression model was performed. Data were analyzed using Statistical Package for Social Sciences (version 14 for Windows; SPSS Inc., Chicago, Ill).

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RESULTS

Copeptin in an animal model of hemorrhagic shock

After the induction of hemorrhagic shock and subsequent reperfusion, copeptin concentrations showed a similar kinetic in all four baboons. Although baboons 118, 137, and 150 had very similar copeptin concentrations at all time points, baboon 157 had a comparable but even more pronounced kinetic (Fig. 1). In all baboons, copeptin increased dramatically from median (range) values of 7.5 (2.7-13) to 157 pM (115-309 pM) after the first hour and continued to increase to 225 pM (214-443 pM) after the second hour. After 3 h of bleeding, before the beginning of reperfusion (time point R0), median (range) copeptin levels reached their maximum with 269 pM (241-456 pM). Copeptin levels dropped after 1 h of reperfusion to 123 pM (87-273 pM) and continued to decline over the next hours until they reached a plateau of 27 (15-78) and 24 pM (13-152 pM) at the end of the experiment.

Fig. 1

Fig. 1

Table 1

Table 1

The MAP followed an inverse kinetic in all animals, decreasing during bleeding and increasing slowly after reperfusion (Fig. 1 and Table 1). The hemorrhage and resuscitation according to this protocol also resulted in metabolic acidosis assessed by base excess and lactate levels (Table 1).

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Copeptin in patients with septic shock

Descriptive characteristics of the patients

The mean age of the 101 patients (55 men and 46 women) was 57 ± 15 (range, 23-86) years. Sepsis was diagnosed in 58% of the patients (on admission in 53 patients [22 with sepsis, 15 with severe sepsis, and 16 with septic shock]; 5 additional patients developed sepsis during their stay in the ICU). Patients fulfilling more than two SIRS criteria were as follows: 99% of 101 patients at admission, 96% of 74 patients on day 2, and 68% of 95 patients on the day of discharge or death. The following percentages of patients were classified as having sepsis, severe sepsis, or septic shock: 53% on admission, 60% on day 2, and 36% on the day of discharge or death, respectively.

On admission, the mean APACHE II score was 22 ± 8 points, and the mean SAPS II score was 53 ± 18 points. The median length of stay in the medical ICU was 4 days (range, 0.2-60 days), and the overall mortality rate was 2 (24). Baseline characteristics with the principal diagnoses of patients are provided in Table 2.

Table 2

Table 2

The principal site of infection was the lung. In 38 (66%) of the 58 patients with infections, the etiologic microorganism was identified, and 14 patients (24%) had bacteremia. The most frequently identified microorganisms were Streptococcus pneumoniae (10.3% of identified microorganisms), Pseudomonas aeurginosa (10.3%), and Escherichia coli (8.6%). Patients with and patients without infection had a similar mortality rate: of the 53 patients admitted with sepsis, severe sepsis, or septic shock, 12 (23%) died of multiorgan failure. Of the 48 patients without infection on admission, 10 (21%) died.

In addition to aggressive fluid repletion, 31% of septic patients needed additional vasoactive treatment with norepinephrine i.v. The mean dose of norepinephrine on admission was 8.7 ± 12.1 μg mL−1; on day 2, 10.1 ± 10.9  μg mL−1; and on the day of discharge/death, 47.2 ± 35.2 μg mL−1 (P < 0.001). Nonsurvivors of severe sepsis and septic shock needed higher doses of norepinephrine as compared with survivors (5.7 ± 7.8 vs. 30.5 ± 28.1 μg mL−1; P < 0.001).

The mean age of the 84 healthy controls was 59 ± 9 years. Controls and patients were well matched regarding age and sex. There was no significant difference of copeptin levels between men and women and no correlation with age.

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Copeptin levels in septic patients as compared with controls

Figure 2A shows copeptin values in healthy blood donors (controls] as compared with critically ill patients with sepsis (i.e., sepsis, severe sepsis, and septic shock) on admission. Median (range) values in controls were 4.1 (1.0- 13.8) as compared with 71.5 (8.5 - 504.0) in patients with sepsis (P < 0.001).

Fig. 2

Fig. 2

In critically ill patients, on admission, there was a stepwise increase of copeptin levels from patients without infection (i.e., SIRS) to patients with sepsis, severe sepsis, and septic shock (Fig. 2B). Thereby, patients with SIRS had median copeptin levels of 27.6 pM (2.3-297 pM), patients with sepsis of 50.0 pM (8.5-268 pM), patients with severe sepsis of 73.6pM (15.3-317 pM), and patients with septic shock of 171.5 pM (35.1-504 pM).

Copeptin levels on admission (in all patients) were correlated with cortisol (r = 0.42; P < 0.001), proatrial natriuretic peptide (r = 0.58; P < 0.001), creatinine (r = 0.60; P < 0.001), and serum osmolality (r = 0.42; P < 0.001). In addition, there was a significant correlation with different parameters of infection (with serum IL-6 levels (r = 0.56; P < 0.001), CRP (r = 0.37; P < 0.001), and ProCT (r = 0.57; P < 0.001)) and with the two physiological scores (APACHE II score (r = 0.49; P < 0.001) and SAPS II score (r = 0.60, P < 0.001)). The correlation with peripheral mean blood pressure was r = −0.21, P < 0.001. There was no correlation with bilirubin level (r = 0.17), glutamic-oxaloacetic transaminase (r = 0.12), glutamic-pyruvic transaminase (r = 0.07), γ-glutamyl transpeptidase (r = 0.04), and alkaline phosphatase (r = −0.08).

Patients with norepinephrine treatment had significantly higher copeptin levels (89.8 [18.5-371] as compared with patients without norepinephrine treatment (50.5 [2.3-504]); P = 0.004). In contrast, patients with corticosteroid treatment had similar copeptin levels (49.5 [7.9-322]) as compared with patients without corticosteroid treatment (61.7 [2.3-504]; P = 0.9).

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Prognostic value of copeptin levels in patients with sepsis, severe sepsis, and septic shock

Figure 3 shows copeptin values in survivors as compared with nonsurvivors with sepsis, severe sepsis, or septic shock measured on admission. Thereby, patients were grouped based on the clinical diagnosis of sepsis according to international guidelines. The median (range) copeptin value on admission in the group of nonsurvivors (144 pM [46.5-504 pM]) was significantly (P = 0.008) higher as compared with the group of survivors (59.1 pM [8.45-386]). This difference between the survivors and nonsurvivors on admission was also significant for IL-6 (P = 0.03) but not for ProCT or CRP (data not shown). In addition, in patients without infections, copeptin values tended to be higher on admission in nonsurvivors as compared with survivors (P = 0.06; 76.1 [15.5-242] as compared with 26.9 [2.3-297])

Fig. 3

Fig. 3

To define the optimal prognostic accuracy for copeptin values in septic patients, we performed ROC analysis, including only data from patients with sepsis, severe sepsis, or septic shock obtained on admission to the ICU. Sensitivity was calculated with those patients who died during their stay on the ICU, and specificity was assessed with those patients who were discharged from the ICU. For comparison, the same ROC analysis was performed with CRP, ProCT, IL-6, SAPS II, and the APACHE II score. The AUC (95% confidence interval (CI)) for copeptin on admission was 0.75 (0.61-0.86). The comparison of the ROC curve AUC of copeptin with the ROC curves of the other parameters (i.e., ProCT (P = 0.5), CRP (P = 0.08), the APACHE II score (P = 0.7), the SAPS II score (P = 0.6), and IL-6 (P = 0.6)) is shown in Table 3.

Table 3

Table 3

The optimal prognostic accuracy for copeptin was 96 pM. At this cut off, which is about 20 times the median of the normal population, the sensitivity was 61.5% and the specificity was 83.8%. In comparison, the APACHE II score, which was also predictive for prognosis, gave similar values as compared with copeptin. At an APACHE II threshold of 27, the sensitivity was 76%, with a specificity of 81.1%.

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Influence of osmolality, creatinine, serum urea, hematocrit, uric acid, and albumin on copeptin levels and outcome

To study potential determinants of copeptin levels, we performed a multiple logistic regression analysis, which included those parameters that correlated significantly with copeptin levels (osmolality, creatinine, hematocrit, urea, uric acid, and albumin). Forward multiple regression with copeptin as dependent and the other parameters as independent factors revealed that creatinine, uric acid, and albumin were significant independent determinants of copeptin levels.

Comparing survivors and nonsurvivors, hematocrit was similar in both groups (33.5% ± 16.9% vs. 34.3% ± 8.3%, P = NS), as were albumin and urea (data not shown). Serum uric acid was significantly higher in nonsurvivors as compared with survivors (513.7 ± 220 vs. 371.3 ± 205 μM; P = 0.004). This was also true for creatinine level (184 ± 133 vs. 111 ± 99 μM; P = 0.008). To evaluate the prognostic value of copeptin and other potential determinants of survival, we performed two multiple logistic regression models, including (1) copeptin, sodium, serum osmolality, uric acid, urea, creatinine, and age, and (2) copeptin, cortisol, and CRP. In both models, serum copeptin levels were the only independent significant predictor of outcome (Table 4, A and B).

Table 4

Table 4

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DISCUSSION

In the present study, we evaluated the concentration of the stable AVP precursor peptide copeptin in hemorrhagic and septic shock. Our findings can be summarized as follows. First, copeptin increased dramatically to more than 30-fold of median normal values after the induction of hemorrhagic shock in baboons and declined immediately after reperfusion of the animals. Second, in critically ill patients, copeptin concentrations gradually increased with the severity of the disease from SIRS over sepsis and severe sepsis to septic shock. Concentrations in septic shock were more than 30-fold higher than in healthy individuals and more than 6-fold higher than in patients with SIRS. Third, copeptin concentrations at the first day of admission to ICU were significantly higher in nonsurvivors than in survivors of septic shock, and the discrimination between the two by ROC plot analysis resulted in similar areas under the curve for copeptin and the physiological APACHE II and SAPS II scores. Fourth, copeptin concentrations were positively correlated to serum osmolality and cortisol concentrations in critically ill patients.

One of the physiological functions of copeptin, the C-terminal part of pro-AVP, is probably its contribution to correct structural formation of AVP before release in the circulation (17). Although further physiological effects may be possible, a practical and relevant property of copeptin is its stoichiometric release together with AVP. Whereas AVP is very difficult to measure, copeptin is stable for days at room temperature (19) and can be detected from 50 μL of plasma or serum within 3 h. The measurement of this very stable AVP precursor fragment can be a clinically relevant and reliable method to substitute the cumbersome assessment of AVP plasma concentration. This concept has also been applied with great success for the A- and B-type natriuretic peptides (28-30) and other difficult-to-measure peptide hormones such as adrenomedullin (31) or endothelin 1 (32).

The measurement of AVP release through copeptin may be of relevance for a variety of clinical situations. Besides the "classical" endocrine indication of diabetes insipidus and syndrome of inappropriate ADH secretion (1, 2, 4, 12, 13), copeptin measurement may also be of relevance in those diseases where electrolyte disturbances, stress response, or cardiovascular instabilities (e.g., shock) influence the vasopressinergic system and contribute to the pathogenesis of the disease.

In this study, we present evidence for this hypothesis. The rapid and up to 35-fold increase of copeptin in our baboon model of hemorrhagic shock is very similar to that of mature AVP as reported in a similar animal model (20). Surprisingly, at the end of hemorrhage, copeptin levels declined rather fast, much faster than expected from the long half-life ex vivo. Again, a similar decline was seen for mature AVP in a similar situation (20). It is possible that hemodilution from autoresuscitation could have played an important role in altering the concentrations of copeptin in this setting. Unfortunately, we did not have hematocrit data for the baboons from these time points that could help identify if this dilution contributed to the decline in copeptin levels.

In an earlier study, we also found a rapid decline of fasting-induced elevated copeptin levels after a water load (19), again a behavior similar to mature AVP. The reason for this discrepancy between fast in vivo decline and long-term ex vivo stability is unknown. Although copeptin may be eliminated via the kidneys (copeptin is detectable in urine; N.G.M., unpublished data) or metabolized in the liver, it is likely that the degradation is due to specific tissue-bound proteases because any proteases present in blood cells or in serum would obviously continue this degradation process ex vivo. It is well described for mature peptide hormones that their plasma concentration is not only controlled through degradation but also by specific receptors, whose function is the binding and internalization of the ligand (e.g., the natriuretic peptide clearance receptor APN-C for the natriuretic peptide family (33)). However, the presence of such a receptor for a precursor fragment such as copeptin is speculative and would suggest further (still unknown) physiological functions that require a tight feedback regulation.

Thus, these observations are of importance for the interpretation of the data. Copeptin does not seem to be only a "junk protein," which may be secreted in a stoichiometric way together with AVP and accumulates subsequently in the circulation, whereas AVP is rapidly eliminated. It is more likely that the kinetics of AVP and copeptin are very similar with respect to increase and decline in vivo. The very stable ex vivo stability of copeptin is independent of the rapid in vivo elimination and, with respect to diagnostics, rather advantageous for the practical usage of this protein.

The release of copeptin as a result of insufficient hemodynamic response is also evident in patients with severe sepsis or septic shock. Sepsis is the leading cause of death in critically ill patients in the United States. It develops in 750,000 people annually, and more than 210,000 of them die (34). Early and adequate diagnosis and risk assessment are pivotal for optimized care of critically ill patients. In an attempt to improve current sepsis definitions, the predisposition, infection, host response, organ dysfunction concept claims for readily measurable circulating biomarkers as an additional tool for the timely assessment and severity classification of septic patients and the prediction of mortality (35). Compared with normal copeptin values, levels in septic shock increased by a factor of 10 to 30 and showed a strong association with the severity of the disease and standard ICU severity assessment systems (such as the APACHE II score).

Patients requiring treatment with norepinephrine had higher copeptin levels than untreated patients. It is unclear if this increase is a direct result of norepinephrine treatment or if it might simply be explained by the association of copeptin and the severity of septic shock. Severely ill patients in septic shock are more likely to receive norepinephrine treatment than septic patients who had not yet progressed to septic shock.

Nonsurvivors in particular had higher copeptin levels as compared with survivors; thus, copeptin levels on admission may be an additional marker of disease severity. This is supported by the fact that in a logistic regression model including other parameters besides copeptin (sodium, serum osmolality, uric acid, urea, albumin, creatinine, age, and cortisol), copeptin levels were the only independent significant predictor of outcome. We are aware that there is a broad overlap between copeptin levels of survivors and nonsurvivors. Fluid status, stress level, and other yet-unrecognized factors may influence copeptin levels. Nevertheless, copeptin may offer additional prognostic information in sepsis, and it is advisable to rely the complex task of prognostic assessment and treatment decisions to several clinical and laboratory parameters, where each may mirror different pathophysiological aspects.

The reported studies on mature AVP in sepsis are heterogenous, and it is presently unclear if patients in septic shock have too much or too little AVP or if initially elevated AVP concentrations may decline during the course of septic shock, leading to a relative insufficiency in later stages (21-23, 36). It is possible that difficulties in AVP measurement may have contributed to discrepant reports, and it will be interesting to see if copeptin measurements may help in the assessment of the role of AVP in septic shock. Particularly in the light of a discussed AVP therapy during septic shock (8, 37-41), copeptin measurement may be of benefit in therapy guidance. Not only is copeptin easier and faster to measure than mature AVP, it is also undisturbed by therapeutically administered AVP, and it would allow the assessment of endogenous AVP production during therapeutic AVP administration.

One shortcoming of this study is that mature AVP was not measured immediately in the patients on ICU. Therefore, a direct comparison between AVP and copeptin in the present cohort of patients was not possible. However, we have reported recently on a very good correlation (r = 0.83) between AVP and copeptin in another set of ICU patients (19). In that report, mature AVP was measured soon after blood withdrawal, whereas copeptin was determined from residual frozen samples after more than 1-year storage time. The present study also drew from a previously collected cohort of frozen samples, and it seems justified to assume a similar correlation between copeptin and AVP. However, the results here need confirmation in a prospective study with immediate measurement of both mature AVP and copeptin. Another shortcoming is that we do not have more detailed information on the fluid status of the patients such as use of diuretics and the exact fluid balance.

In conclusion, we describe elevated levels of the stable C-terminal AVP precursor copeptin in hemorrhagic and septic shock together with an association of these elevated levels to the outcome of the patients. If confirmed in prospective studies, copeptin may be a useful surrogate marker for fast AVP measurement in a variety of diseases.

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ACKNOWLEDGMENTS

The authors thank the staff of the medical intensive care unit, Prof. Rudolf Ritz, the laboratory of chemical pathology of the University Hospital Basel, Prof. Peter Huber and Dr. Marc A. Viollier, and Johanna Hetzel, Anne Schmiedel, Barbara Schäffus, Uwe Zingler, and Frank Bonconseuil at B R A H M S AG for excellent technical assistance.

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Keywords:

AVP; ADH; sepsis; hormone precursor; copeptin

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