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).
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.
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.
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).
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).
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])
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.
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%.
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).
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.
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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AVP; ADH; sepsis; hormone precursor; copeptin