Arab. J. Lab. Med. (2014) Vol. 40, No. 1 : 729 - 742

COPEPTIN, A NOVEL NEUROPEPTIDE IN ACUTE MYOCARDIAL INFARCTION

GAMIL ABDALLA, AHMED ELSHAFEI, OSSAMA ABD EL-MOTAAL, TAREK SALMAN* and ABD EL-RAHMAN ELBOQUIRY**

*Biochemistry and Molecular Biology Department, Faculty of Pharmacy (Boys), Al-Azhar University, Nasser City, Cairo, Egypt.

**Cardiac Catheterization Unit, National Heart Institute, Imbaba, Giza, Egypt.

Abstract:

Aim of study: To investigate the diagnostic role of copeptin, a novel neuropeptide derived from pre-

proarginine vasopressin (preproAVP), as a messenger of stress signals in early identification of acute

myocardial infarction (AMI) within early hours of admission into emergency department (ED), alone

and as combination with other well established necrotic biomarkers as; cardiac troponin-I (cTnI) and

myocardial band of creatine kinase (CK-MB).

Subjects and Methods: This study was performed on 50 subjects admitted to ED of the National

Heart Institute (NHI) suffering from acute chest pain within 3 hours from chest pain onset. Patients

with positive electrocardiographic (ECG) evidences were excluded. They were divided into 33 pa-

tients with AMI and 17 patients with unstable angina (UA). Concentrations of copeptin, cTnI and

CK-MB were determined in sera of the individuals at the time of admission, 3 and 6 hours later.

Copeptin and cTnI were determined using commercially available enzyme linked immunosorbent

assay (ELISA) kits, while CK-MB was determined spectrophotometrically.

Results: Copeptin’s serum level at three hours after admission to ED was significantly higher than its

level at admission time and after 6 hours (P < 0.001) in AMI group, while serum level of both cTnI

and CKMB exhibited a gradual increase with time to reach the peak value at 6 hours (P < 0.001 for

both). Regarding UA group, serum copeptin didn’t show significance of difference between the three

samples, while serum cTnI and CKMB were elevated significantly as time evolves. Different cutoff

values were defined for each biomarker at each time of assessment. At admission time, The sensi-

tivity and specificity of copeptin and cTn-I combination were 100% and 100% respectively, versus

93.9% and 94.1% for copeptin only, also 72.7% and 82.4% for cTnI alone, as well as versus 66.7%

and 70.6 for CK-MB. cTnI and CK-MB combination showed sensitivity and specificity of 97% and

94.1%. The area under curve (AUC) of the cTnI-copeptin combination was 1 which was significantly

higher than the AUC of cTnI alone (0.81) and that of cTnI-CK-MB combination (0.92).

Conclusion: Copeptin as a marker of acute stress together with the cardiospecific cTnI represent a

promising diagnostic approach for accelerated early identification of AMI patients. In a cutoff value

dependent manner, copeptin-cTnI combination assay at time of admission for patients with recent

onset of chest pain (3 hours) was significantly superior to the use of conventional cTnI assay in the

diagnostic accuracy.

730

Introduction:

Ischemic heart diseases (IHD) are the com-monest variety of cardiovascular diseases; include a wide spectrum of conditions ranging from silent angina, UA, AMI, heart failure and sudden death (1). ST-segment elevated AMI (STEMI), Non ST-ele-vated AMI (NSTEMI) and UA represent the acute coronary syndrome (ACS) (2). IHD are the leading cause of death in high and middle income countries and the 4th cause of death in low income countries. Globally, 7.3 million people died from IHD in 2011. STEMI occurs in 3 million, while NSTEMI in 4 million people approximately (3).

The main pathogenic mechanism underly-ing IHD is the integration between atherosclerotic plaque rupturing and intravascular thrombosis, which is influenced by a complex interaction be-tween procoagulants, anticoagulant, fibrinoloytic, endothelial dysfunction and atherosclerosis inflam-matory processes (4-6).

From the pathological view, AMI (heart attack) is defined as myocardial death and necrosis due to prolonged ischemia (7, 8). According to the latest diagnostic criteria for AMI, the clinical picture to-gether with ECG findings and elevation of the most specific myocardial necrotic biomarker, cTnI are the standard diagnostic tool to identify AMI patients (8).

The shift of stable IHD into unstable state, UA develops. This occurs when coronary circulation becomes reduced due to atherosclerotic plaque rup-ture and thrombus formation. UA can predispose NSTEMI and STEMI if untreated appropriately (9, 10). STEMI is a clinical type of AMI characterized by elevation of ST segment in the ECG as a frank sign for diagnosis, while UA and NSTEMI are tightly closed conditions common in their suggestive isch-emic symptoms and lack of ECG diagnostic signs. However, distinction between UA and NSTEMI is

made by the presence or absence of cardiospecific necrotic biomarkers (2). For patients identified early with NSTEMI, immediate catheterization for revas-cularization of the unstable coronary lesions may prevent development of ischemic complications that would develop during therapeutic intervention. Intensive antiplatelets therapy before PCI may re-duce unstable lesions thrombotic burden and aug-ments safety and successfulness of PCI (11).

Biomarkers are useful in the evaluation of chest pain patients when they are highly sensitive to safely “rule out” cardiac ischemia or when they are highly specific to capture patients with ACS who otherwise have non-diagnostic tests (e.g. ECG). Therefore, cTns have become a prominent role in the diag-nosis of AMI. Cardiac troponins (cTns), sensitive and specific biochemical markers of cardiomyo-cyte necrosis, are very helpful in clinical practice to identify patients with acute coronary syndromes at high risk (8,13). The major limitation of conventional cTns assay is sensitivity deficit at presentation, due to its delayed release from the necrotized myocytes and plasma level elevation. Exclusion of AMI con-sequently requires prolonged monitoring over 6-9 hours and serial blood sampling (14-17). If cTns assay is not available, the best alternative is CK-MB (8).

Regarding the previously discussed challenges for early identification of AMI and early medical intervention, we aimed in this study to investigate the possibility of using pathophysiologically differ-ent biomarkers in their release pattern to accelerate early clinical recognition of AMI patients or ruling out AMI for patients of other causes of chest pain as single or combined biomarkers. The investigated biomarkers were; cTns and CK-MB as myocardial necrotic biomarkers and copeptin as a surrogate bio-marker for AVP mediated stress adaptation axis.

Arginine vasopressin (AVP), also described as

731

the antidiuretic hormone (ADH), is a nona cyclic peptide with intramolecular disulfide bonds be-tween Cys4 and Cys9. It is a hormone with impor-tant osmoregulatory, hemodynamic, hemostatic, neuroendocrine and central nervous effects (18-20). It is stored and released from the magnocellular nerve endings in the posterior pituitary gland in response to changes in blood volume, water osmolality and other hemodynamic changes (20). However, it is re-leased from the parvocellular nerve terminals into the portal circulation to affect the corticotrophic cells in the adenohypophysis in synchronism with corticotropin releasing hormone (CRH) as response to stress states (21). AVP acts on three main receptors, where it mediates antidiuretic effects, strong arterio-lar vasoconstriction and adrenocorticotropic hormone (ACTH) secretion during stress response (22). AVP is an end product of its ancestor polypeptide precur-sor, prepro-AVP. Upon cleavage of this ancestor preproprotein, AVP will be released in conjunction with neurophysin-II and copeptin (23).

Copeptin is 39 amino acids glycopeptide with molecular weight of 5000 KD representing the c-terminal segment of prepro-AVP and stoichiometri-cally released with AVP in response to stimuli af-fecting plasma AVP level (24-27). It was firstly defined by Holwerd in 1972 (28). It is considered as a novel neurohormone of the AVP system (29, 30). Copeptin’s physiological function is not well-established, but it was hypothesized that copeptin is essential for proper proteolytic maturation, folding and struc-tural stability of pro-AVP through interaction with calnexin-calreticulin chaperone system (18, 20, 30, 31). It is more stable than AVP invitro, easier in assay and has reliable measuring methods. So, it replaced the time wasting AVP assay cumbersome methods in clinical analysis (26).

In 2008, Katan and his colleagues reported signif-icant positive correlation between plasma copeptin

and individual stress level with much more stability of samples relative to cortisol that has physiologi-cal diurnal variation in plasma level in addition to plasma cortisol assay challenges (32). The vasopress-inergic/CRH system has a significant role in condi-tioning to chronic stress as in obesity and diabetes. Recently, it is observed that, plasma copeptin level positively correlates to states of insulin resistance, diabetes mellitus, obesity, metabolic syndrome and diabetic nephropathy (33-35). During acute illness, acute stress usually develops with high magnitude of plasma copeptin as a neuropeptide of the AVP/CRH system (36). These findings promoted copeptin introduction as diagnostic and prognostic biomarker in acute medicine (37).

Subjects and Methods:

This study enrolled 50 subjects admitted to the ED of NHI with symptoms suggestive for ACS within 3 hours from chest pain onset. Patients with postivie ECG findings (STEMI) were excluded. Ac-cording to clinical features, serum biomarkers lev-els and clinical decision, they were divided into two groups:

1-AMI group comprised 33 diagnosed as having AMI of age (47-73 years) with mean ± SEM (58.9 ± 1.11 years) and BMI of range (23.18-39.06 Kg/m2) with mean ± SEM (30.2 ± 0.75 Kg/m2), 22 were males and 11 were females.

2-UA group comprised 17 patients diagnosed as having UA of age (49-71 years) with mean ± SEM (59.2 ± 1.46 years) and BMI of range (23.53-38.95 Kg/m2) with mean ± SEM (30.6 ± 1.08 Kg/m2), 11 were males and 6 were females.

Venous blood samples were withdrawn from the admitted patients three times in the following inter-vals, at zero time (the time of admission), after 3 and 6 hours. The samples were collected in serum separator tubes (BD vacutainer system) and allowed

732

to clot for 15 minutes, and then centrifuged at 4000 rpm for 10 minutes. Sera were separated and divid-ed into aliquots and stored at -80˚C until the time of analysis.

Serum copeptin and cTnI were quantitatively measured using their commercially available ELI-SA technique based kits supplied by Phoenix Phar-maceuticals, inc: USA, and Monobind Inc: USA, re-spectively, according to the principle of (Porstmann and Kiessig, 1992) (38) for copeptin and (Apple et al., 1999) (39) for cTnI. CK-MB was spectrophoto-metrically measured in quantitative manner using photometer 5010 V5+, Germany using kits supplied by Stanbio: USA, according to principle of (Daw-son et al., 1965) (40).

Results:

Graph pad prism program version 5.0 and Mac Apple Epi-Stat S3A Pro statistics package (V. 4.0, Apple corporation, USA, 2012) were used for data drafting and analysis. Data was summarized as mean ± SEM. For two quantitative data analysis, paired, non-parametric Wilcoxon test was performed, while non-parametric Fried-man’s test was used for analy-sis of more than two quantitative data followed by Dunns for detection of significance. Significance was considered when P value < 0.05.

Diagnostic validity test was done including: di-agnostic specificity, sensitivity, positive predictive value (PPV) and negative predictive value (NPV). The receiver operator characteristic (ROC) curve was constructed to obtain the most sensitive and specific cutoff value for each biomarker. To evalu-ate the most discriminating markers between the compared groups, area under ROC curve can also be calculated. Multi-ROC or combination between more than 1 parameter was used to improve sen-sitivity or specificity or both using 2 best cutoffs together.

In AMI group, serum copeptin showed its peak value at three hours from admission, which was sig-nificantly higher than admission time and at 6 hours (P < 0.001), while both cTnI and CK-MB serum levels gradually increased in proportion with time to reach their maximum values at 6 hours samples than 3 hours and admission samples (P < 0.001) (Table-1) (Figure-1).

Serum copeptin didn’t show significant differ-ences between its levels at any assay time in UA group, in contrast to both cTnI and CK-MB which did show significant gradual increase in their serum level with time, but didn’t reach the diagnostic cut-off values (Table-2 and figure-2).

The representative time courses and release pat-tern of copeptin, cTnI and CK-MB in both AMI and UA groups are demonstrated in figure-3. As what ap-pear in this figure, in AMI group, copeptin showed proportional increase till reaching the 3 hours post admission sample, when it reached its maximal val-ue after which it declined (P < 0.001), while both cTnI and CK-MB levels showed continuous in-crease to reach their maximal level at 6 hours (P < 0.001). In UA group, copeptin remained unchanged within 6 hours after admission, while cTnI and CK-MB serum levels continuously increased within 6 hours, but so far values than that of AMI.

Cutoff values of copeptin, cTnI and CK-MB with their respective sensitivity, specificity, NPV and PPV, in addition to their corresponding AUC from their ROC curves at admission time and 3 hours after are illustrated in table-3 and figure-4.

Multi ROC curves for copeptin-cTnI and CK-MB-cTnI combinations at admission time and three hours later are illustrated in figure-5 and their cor-responding cutoff values and AUC are listed in ta-bles-4, 5, 6 and 7.

733

Table (1): The mean (± SEM) serum levels of copeptin, cTnI and CK-MB in AMI group.

Table (2): The mean (± SEM) serum levels of copeptin, cTnI and CK-MB in UA group.

Table (3): The cutoff value, sensitivity, specificity, NPV, PPV and AUC of copeptin, cTnI and CK-MB at admission time and 3 hours later.

Table (4): The diagnostic validity of combined ROC curve for copeptin at cutoff value of 30.7 pmol/L and cTnI at cutoff value of 0.58 ng/mLat admission time.

Table (5): The diagnostic validity of combined ROC curve for cTnI at cutoff value of 0.87 ng/mL and CK-MB at cutoff value of 18.0 U/L at admission time.

Table (6): The diagnostic validity of combined ROC curve for copeptin at cutoff value of 51.7 pmol/L and cTnI at cutoff value of 1.01 ng/mLat admission time.

Table (7): The diagnostic validity of combined ROC curve for cTnI at cutoff value of 1.02 ng/mL and CK-MB at cutoff value of 146 U/L at admission time.

734

Figure-1. The mean (± SEM) serum levels of copeptin, cTnI and CK-MB in AMI group.

Figure-2. The mean (± SEM) serum levels of copeptin, cTnI and CK-MB in UA group.

735

Figure (3) : The representative time course and release pattern of copeptin, cTnI and CK-MB in AMI and UA groups during the first six hours after admission.

Figure (4) : The ROC curves of copeptin, cTnI and CK-MB at admission time (A) and 3 hours later (B).

Figure (5) : Multi ROC curves of copeptin-cTnI and cTnI-CK-MB combinations at admission time (A) and three hours later (B).

736

Discussion:

In the present study the mean serum level of co-peptin was highly significant in three hours of AMI group (89.5 ± 5.93 pmol/L) than the admission time and six hours (54 ± 2.13 pmol/L and 60.9 ± 4.19 pmol/L respectively) at P < 0.001.

Two hypotheses may demonstrate why plasma copeptin was elevated within the early three hours in AMI group. The hypothesis of stress represents the first one, where plasma copeptin/AVP level was raised as a substantial part of the endocrine stress response resulting in synergistic release of ACTH and cortisol (15, 21, 22, 26, 30, 41). Considering AMI a life threatening stressor, it gives an input to the brain stem and limbic system where emotional responses are controlled, which in turn stimulate the parvocel-lular neurons of the hypothalamic SON to release both AVP and CRH into the portal circulation to af-fect the anterior pituitary to release the ACTH from the corticotrophic cells in synergistic manner, which consequently stimulates adrenal cortisol synthesis and release (37, 42, 43).

The second hypothesis is the hemodynamic hy-pothesis, where acute changes in cardiac dynam-ics, cardiac under filling, and stimulation of cardiac baroreceptors as a response for reduced systemic blood pressure or direct baroreceptors damage re-sulting from prolonged ischemia represent a strik-ing trigger for stimulation of AVP/copeptin release (21, 22, 26, 41, 44).

The decline in plasma copeptin afterward is questionable in light of its extreme invitro stabil-ity. It may be a result of its short invivo half-life as its shadow, AVP. Morgenthaler et al., 2006 found that, plasma copeptin level behaves like AVP in its kinetics, where its plasma half-life declines quickly with unknown plasma clearance mechanism. Also,

it may be due to the ischemic compensatory mecha-nisms, such as initiation of arteriogenesis of the collateral coronary arteries, which may reduce the ischemic symptoms, decrease cardiac baroreceptors stimulation and consequently reduce copeptin/AVP release axis. Another proposal may originate from reduction of stress firing acuity with time after the onset of chest pain (25).

These results were in accordance with Reichlin et al., 2009 (14), Keller et al., 2010 (15), Meune et al., 2011 (45), Charpentier et al., 2012 (46), Folli et al., 2013 (47) and Maisel et al., 2013 (41) where copeptin levels at admission were high in AMI group present-ing zero to four hours after onset of symptoms with a falling pattern afterward from five to ten hours.

cTns are structural and regulatory proteins of skeletal and cardiac muscle cells and are of essential importance in the regulation of muscle cell contrac-tion (48). Regarding the latest third universal defini-tion of AMI, cTns are considered the gold standards in diagnosis of myocardial necrosis (8, 23).

The mean serum level of cTnI was highly sig-nificant in six hours of AMI group (12 ± 0.83 ng/mL) than the admission time and three hours (0.89 ± 0.03 ng/mL and 1.19 ± 0.02 ng/mL respectively) at P < 0.001.

In relation to cTnI, White, 2011 suggested six potential pathobilogical mechanisms underlying cTns elevations: 1) Myocyte necrosis, 2) apopto-sis, 3) normal myocyte turnover, 4) cellular release of proteolytic troponin degradation products, 5) increased cell membrane permeability and 6) for-mation and release of membranous blebs (49). Re-garding this pathological conditions, myocardial necrosis appears to be the main cause of serum cTnI elevation. To explain the gradual release of cTnI and subsequent elevation in its plasma level, the

737

intracellular location of cTnI should be considered. Approximately 5-8% of cTnI is free (unbound) in the cytosol, while the rest is incorporated in the con-tractile apparatus of the cell (50, 51). So, when cardio-myocytes become necrotized, the cytosolic pool is released first followed by a more protracted release from cTnI stores due to the continuous degradation of the contractile apparatus and release of cTnI. In accordance with these results a studies were car-ried out by Charpentier et al., 2012 (46), Chenevier-Gobeaux et al., 2013 (52) and Maisel et al., 2013 (41) where cTnI level showed a delayed increase after admission of patients with AMI.

CK-MB is an isoform of CK that is predomi-nantly located in myocardial cells and is released into the circulation in the setting of MI (53). In this study the mean serum level of CK-MB was highly significant in three and six hours of AMI group (199 ± 16.4 U/L and 369 ± 37.1 U/L respectively) than the admission time (73.8 ± 10.1 U/L) at P < 0.001. Also the mean serum level of CK-MB was highly significant in six hours (369 ± 37.1 U/L) than the three hour (199 ± 16.4 U/L) at P < 0.001.

The gradual increase in level of CK-MB in AMI group in this study may be due to its location pre-dominantly in cytoplasmic pool of myocardial cells. Therefore, after disruption of the sarcolemma of the cardiomyocyte, the cytoplasmic pool of CK-MB is released first and continues rising as ischemia pro-longed, resulting in more cardiomyocytes necrosis. These results agree with a study carried out by Es-ses et al., 2001 (54) where CK-MB was measured on admission, six, twelve, eighteen in which a gradual increase in its level in MI group.

Regarding the mean serum copeptin value in UA group, a non-significant difference was found be-tween the admission time, three and six hours (12.7 ± 2.96 pmol/L, 16.9 ± 5.43 pmol/L and 12.5 ± 2.93

pmol/L respectively). These results may be due to insufficient stress stimulus to aggravate copeptin/AVP release and HPA axis activation. It may be also due to absence of direct damage for cardiac baro-receptors or occurrence of cardiac hemodynamic changes, because of the incomplete occlusion of coronary arteries. In accordance to these results a study carried out by Reichlin et al., 2009 (14), Keller et al., 2010 (15), Charpentier et al., 2012 (46), Folli et al., 2013 (47) and Maisel et al., 2013 (41) in which the copeptin values of UA subset of patients with ACS were normal and didn’t show differences from those observed in patients with other causes of chest pain.

The mean serum level of cTnI (ng/mL) was highly significant in three and six hours of UA group (0.86 ± 0.06 ng/mL and 1.01 ± 0.05 ng/mL respectively) than the admission time (0.58 ± 0.06 ng/mL) at P < 0.001, also

the mean serum level of cTnI was highly signifi-cant in six hours (1.01 ± 0.05 ng/mL) than the three hour (0.86 ± 0.06 ng/mL) at P < 0.001.

In UA, there is no trigger for cTnI release as the cardiac myocyte still intact without any pathologi-cal necrosis, but the presence of minute amounts of serum cTnI in UA group may result from: the ischemic reversible injury to the cardiomyocytes and myocardial stretching as in UA increase cardio-myocytes membranes permeability to cytoplasmic cTnI (55). In addition to this mechanism, cTnI may increase as a result for its intracellular proteolysis to yield small fragments that can cross an intact cell membrane. These proteolytic fragments were observed after 15 minutes of mild ischemia (56, 57). Another mechanism that may clarify the cause of cTnI increase originates from the active secreting vesicles (blebs) concept. This concept hypothesized that, during myocardial hypoxia as in UA, cTnI is packaged and released a cross the intact cell mem-

738

brane within secretory vesicles, but this hypothesis needs further studies to augment its fact (49, 58). However, cTnI values in UA group didn’t reach AMI diagnostic cutoff value at different time inter-vals.

Regarding the mean serum level of CK-MB was highly significant in three and six hours of UA group (126 ± 19.8 U/L and 189 ± 28.3 U/L respec-tively) than the admission time (32 ± 5.75 U/L) at P < 0.001 also the mean serum level of CK-MB was highly significant in six hours (189 ± 28.3 U/L) than the three hour (126 ± 19.8 U/L) at P < 0.001. The increase in serum CK-MB level is not entirely specific for AMI, but may reflect some degree of ischemic heart damage as in UA (59, 60). Owing to the reversible myocardial damage with UA and the cy-toplasmic location of CK-MB, it is not a surprise to be slightly elevated in this group. From another view, ischemia alone may be the cause of CK-MB elevation without cell injury as what was observed by Ishikawa et al., 1997 (61).

Early identification of patients at risk in a popu-lation with undifferentiated chest pain is essential since these patients need an aggressive therapeutic regimen (15, 41).

At admission time; cTnI with cutoff value of 0.87 ng/mL revealed AUC = 0.81, with sensitivity 72.7%, specificity 82.4%, NPV 60.9% and PPV 88.9%, while copeptin diagnostic accuracy at the same time at cutoff value of 30.7 pmol/L was much higher than that of cTnI with AUC = 0.98, with sen-sitivity 93.9%, specificity 94.1%, NPV 88.9%, PPV 96.9%, so copeptin was more sensitive and specific than cTnI with better NPV and PPV. These results were in accordance with Keller et al., 2010 (15)and Ray et al., 2012 (62).

Using the dual marker strategy, involving the

different pathophysiological basis of release of cTnI as the most specific biomarker for cardiomyocytes injury and copeptin as an indicator for acute stress and hemodynamic instability; theoretically it would provide more accurate diagnostic performance.

When both ROC curves of cTnI and copeptin were merged together, a positive impact on diag-nostic performance was improved reaching AUC of 1.00, sensitivity 100%, Specificity 100%, NPV 100% and PPV 100% at cutoff value for copeptin 30.7 pmol/L and cut-off value for cTnI 0.58 ng/mL. Hence, addition of copeptin to cTnI for early iden-tification of AMI patients enlarges cTnI AUC and improves diagnostic accuracy. These results were in accordance with Reichlin et al., 2009 (14), Keller et al., 2010 (15), Charpentier et al., 2012 (46), Ray et al., 2012 (62) and Maisel et al., 2013 (41).

At the admission time; CK-MB with cutoff value of 32 U/L revealed AUC = 0.77, with sen-sitivity 66.7%, specificity 70.6%, NPV 52.2% and PPV 81.5%. However, upon combination of both cTnI and CK-MB at admission time, the AUC was 0.92 with sensitivity 97%, specificity 94.1%, NPV 94.1% and PPV 97% at cutoff value for cTnI 0.87 ng/mL and for CK-MB 18 U/L. In spite of the obvi-ous added diagnostic value of CK-MB to cTnI at admission time; but it was still lower than that of copeptin and cTnI combination.

At three hours; cTnI with cutoff value of 1.02 ng/mL revealed AUC = 0.93, with sensitivity 90.9%, specificity 64.7, NPV 78.6% and PPV 83.3%, while copeptin diagnostic accuracy at the same time at cutoff value of 51.7 pmol/L was much higher than that of cTnI with AUC = 0.97, with sensitivity 97%, specificity 88.2%, NPV 93.8%, PPV 94.1%, so co-peptin was more sensitive and specific than cTnI with better NPV and PPV. When both ROC curves of cTnI and copeptin were combined together at

739

three hours, a positive impact on diagnostic perfor-mance was improved reaching AUC of 1.00, sensi-tivity 100%, Specificity 100%, NPV 100% and PPV 100% at cutoff value for copeptin 51.7 pmol/L and cut-off value for cTnI 1.01 ng/mL. Hence, addition of copeptin to cTnI for early identification of AMI patients enlarges cTnI AUC and improves diagnos-tic accuracy. These results were in accordance with Reichlin et al., 2009 (14), Keller et al., 2010 (15).

At the three hours; CK-MB with cutoff value of 146 U/L revealed AUC = 0.73, with sensitivity 69.7%, specificity 64.7%, NPV 52.4% and PPV 79.3%. However upon combination of both cTnI and CK-MB at three hours, the AUC was 0.97 with sensitivity 97%, specificity 100%, NPV 94.4% and PPV 100% at cutoff value for cTnI 1.02 ng/mL and for CK-MB 146 U/L. This combination provides a higher specificity and PPV than that at admission time. In spite of the obvious added diagnostic value of CK-MB to cTnI at three hours; but it was still lower than that of copeptin and cTnI combination.

In conclusion, copeptin as a more stable and easier in analysis mirror for AVP opened a door of investigating vasopressinergic system involvement in many stress implicated medical diseases. In AMI, copeptin exhibited a superior diagnostic value as a single biomarker in comparison to the standard biomarker, cTns for early detection of NSTEMI within the early 3 hours from chest pain onset, con-sidering its lower specificity in comparsion to cTnI. This study reported that, the use of dual biomark-ers approach of different release pattern as cTnI and copeptin showed higher diagnostic value in early detection of AMI than using cTnI alone, CK-MB alone or cTnI-CK-MB combination.

References:

1. Anantharaman V and Lim S. (2013): Treatment of NSTE-MI (Non-ST Elevation Myocardial Infarction). Curr

Emerg Hosp Med Rep. 1:18-28.

2. Boden H, van der Hoeven L, Karalis I, Schalij J and Juke-ma W. (2012): Management of acute coronary syndrome: achievements and goals still to pursue. Novel develop-ments in diagnosis and treatment. J Intern Med. 271:521-536.

3. World health organization (2011). The top ten causes of death. Fact sheet No 310.

4. Ahmad M and Sharma N. (2012): Biomarkers in acute myocardial infarction. J Clin Exp Cardiolog. 3:11.

5. Badimon L, Padró T and Vilahur G. (2012): Atherosclero-sis, platelets and thrombosis in acute ischemic heart dis-ease. Eur Heart J: Acute Cardiovasc Care. 1: 60-74.

6. Saffitz J. (2012): The heart. In Rubin E. Rubin’s pathol-ogy: Clinicopathologic foundations of medicine.6th ed. Philadelphia. Lippincott Williams & Wilkins, a Wolters Kluwer business.

7. Schoen F and Mitchell R. (2010): The heart. In kumar V, Abbas A, Fausto N and Aster J. Robbins and Cotran patho-logical basis of disease.8th ed. Philadelphia. Saunders, an imprint of Elsevier Inc.

8. Thygesen K, Alpert J, Jaffe A, Simoons M, Chaitman B, and White H: the Writing Group on behalf of the Joint ESC/ACCF/AHA/WHF Task Force for the Universal Definition of Myocardial Infarction. (2012): Third inter-national definition of myocardial infarction. Circulation. 126:2020-2035.

9. Leeper B, Cyr A, Lambert C and Martin K. (2011): Acute coronary syndrome. Crit Care Nurs Clin North Am. 23(4):547-557.

10. Cannon C and Braunwald E (2012): Unstable Angina and Non-ST Elevation Myocardial Infarction. In: Bonow R, Mann D, Zipes D and Libby P. Braunwald’s heart disease; a textbook of cardiovascular medicine. 9th ed. Philadel-phia. Saunders, an imprint of Elsevier Inc.

11. Jneid H, Anderson J, Wright R, Adams C, Bridges C, Casey D, et al. (2012): American College of Cardiology Foundation; American Heart Association Task Force on Practice Guidelines. 2012 ACCF/AHA focused update of the guideline for the management of patients with unstable angina/Non-ST-elevation myocardial infarction (updat-ing the 2007 guideline and replacing the 2011 focused

740update): a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines. Circulation. 126(7):875-910.

12. Lin S, Yokoyama H, Rac V and Brooks S. (2012): Novel biomarkers in diagnosing cardiac ischemia in the emer-gency department: a systematic review. Resuscitation. 83(6):684-691.

13. Mueller C. (2012): Detection of myocardial infarction-Is it all troponin? Role of new markers. Clin Chem. 58 (1): 162-164.

14. Reichlin T, Hochholzer W, Stelzig C, Laule K, Freidank H, Morgenthale N, et al. (2009): Incremental value of co-peptin for rapid role out of acute myocardial infarction. J Am Coll Cardiol. 54:60-68.

15. Keller T, Tzikas S, Zeller T, Czyz E, Lillpopp L, Ojeda M, et al. (2010): Copeptin improves early diagnosis of acute myocardial infarction. J Am Coll Cardiol. 55(19):2096-2106.

16. Thygesen K, Mair J, Katus H, Plebani M, Venge P, Collin-son P, et al. (2010): Study Group on Biomarkers in Cardi-ology of the ESC Working Group on Acute Cardiac Care. Recommendations for the use of cardiac troponin mea-surement in acute cardiac care. Eur Heart J. 31(18):2197-2204.

17. Gu Y, Voors A, Zijlstra F, Hillege H, Struck J, Masson S, et al. (2011): Comparison of the temporal release pattern of copeptin with conventional biomarkers in acute myocar-dial infarction. Clin Res Cardiol. 100:1069-1076.

18. Morgenthaler N, Struck J, Jochberger S and Duser W. (2008): Copeptin: clinical use of new biomarker. Trends Endocrinol Metabol. 19:43-49.

19. Vincent J and Su F. (2008). Physiology and pathophysiol-ogy of the vasopressinergic system. Best Pract Res Clin Anaesthesiol. 22(2):243-252.

20. Brunton L, Chabner B and Knollman B. (2011): Goodman and Gilamn’s the pharmacological basis of therapeutics. 12th ed. USA. McGrow-Hill companies.

21. Morgenthaler N. (2010): Copeptin: A biomarker of car-diovascular and renal function. Congest Heart Fail. 16(4) (s1):S37-S44.

22. Lippi G, Plebani M, Di Somma S, Monzani V, Tubaro

M, Volpe M, et al. (2012): Considerations for early acute myocardial infarction rule-out for emergency department chest pain patients: the case of copeptin. Clin Chem Lab Med. 50(2):243-253.

23. Aldous S. (2013): Cardiac biomarkers in acute myocardial infarction. Inter J Cardiol. 164(3):282-294.

24. Struck J, Morgenthaler N and Bergmann A. (2005): Co-peptin, a stable peptide derived from the vasopressin pre-cursor, is elevated in serum of sepsis patients. Peptides. 26:2500-2504.

25. Morgenthaler N, Struck J, Alonso C and Bergmann A. (2006): Assay for the measurement of copeptin, a stable peptide derived from the precursor of vasopressin. Clin Chem. 52(1):112-119.

26. Hammarsten O and Goetze P. (2012): Copeptin: A new peptide in clinical measurement. Kilnisk Niokemi I Nor-den. (1): 22-29.

27. Mastropietro W, Mahan. M, Valentine M, Clark A, Hines C, Walters III L, et al. (2012): Copeptin as a marker of relative arginine vasopressin deficiency after pediatric car-diac surgery. Intensive Care Med. 38:2047-2054.

28. Holwerda D. (1972): A glycopeptide from the posterior lobe of pig pituitaries. I. Isolation and characterization. Eur J Biochem. 28: 334-339.

29. Yalta K, Sivri N, Yalta T, Geyik B, Aksoy Y and Yetkin E. (2011): Copeptin (C-terminal provasopressin): a promis-ing marker of arrhythmogenesis in arrhythmia prone sub-jects? Int J Cardiol. 148(1):105.

30. Yalta K, Yalta T, Sirvi N and Yetkin E. (2013): Copeptin and cardiovascular disease: A review of a novel neurohor-mone. Inter J Cardiol. 167:1750-1759.

31. Bhandari S, Loke I, Davies J, Squire I, Struck J and Ng L. (2009): Gender and renal function influence plasma levels of copeptin in healthy individuals. Clin Sci. 116:257-263.

32. Katan M, Morgenthaler N, Widmer I, Puder J, König C, Müller B et al. (2008): Copeptin, a stable peptide derived from the vasopressin precursor, correlates with the indi-vidual stress level. Neuro Endocrinol Lett. 29(3):341-346.

33. Enhörning S, Wang T, Nilsson P, Almgren P, Hedblad B, Berglund G, et al. (2010): Plasma copeptin and the risk of diabetes mellitus. Circulation. 121(19):2102-2108.

74134. Enhörning S, Struck J, Wirfält E, Hedblad B, Morgenthal-

er N and Melander O. (2011): Plasma copeptin, a unifying factor behind the metabolic syndrome. J Clin Endocrinol Metab. 96(7):1065-1072.

35. Enhörning S, Bankir L, Bouby N, Struck J, Hedblad B, Persson M, et al. (2013): Copeptin, a marker of vasopres-sin in abdominal obesity, diabetes and microalbuminuria: the prospective Malmö Diet and Cancer Study cardiovas-cular cohort. Int J Obes. 37(4):598-603.

36. Katan M and Christ-Crain M. (2010): The stress hormone copeptin: A new prognostic biomarker in acute illness. Swiss Med Wkly. 140:w13101.

37. Nickel C, Bingisser R and Morgenthaler N. (2012): The role of copeptin as a diagnostic and prognostic biomarker for risk stratification in the emergency department. BMC Med. 10:7.

38. Porstmann T and Kiessig S. (1992): Enzyme immunoas-say techniques. An overview. J Immunol Methods. 150(1-2):5-21.

39. Apple F, Christenson R, Valdes R, Andriak A, Duh Show-Hong, Feng Y, et al. (1999): Simultaneous rapid measure-ment of whole blood Myoglobin, Creatine Kinase-MB and Cardiac Troponin-I by the Triage cardiac panel for detec-tion of myocardial infarction. Clin Chem. 45: 199-205.

40. Dawson D, Eppenberger H and Kaplan N. (1965): Cre-atine Kinase evidence for a dimeric structure. Biochem biophys Res Commun. 21:346-353.

41. Maisel A, Mueller C, Neath S, Christenson R, Morgen-thaler N, McCord J, et al. (2013): Copeptin helps in the early detection of patients with acute myocardial infarc-tion. J Am Coll Cardiol. 62(2):150-160.

42. Kyrou I and Tsigos C. (2009): Stress hormones: Physi-ological stress and regulation of metabolism. Curr Opin Pharmacol. 9(6):787-793.

43. Aguilera G. (2011): HPA axis responsiveness to stress: im-plications for healthy aging. Exp Gerontol. 46(2-3):90-95.

44. Jerichow T, Struck J, Vollert L, Vogt B, Mans D, Schro-der M, et al. (2011): Elevation of plasma copeptin in acute myocardial infarction in pigs is related to changes in mean arterial blood pressure but not to myocardial ischemia. J Am Coll Cardiol. 57 (14); E925.

45. Meune C, Zuily S, Wahbi K, Claessens Y, Weber S and Chenevier-Gobeaux C. (2011): Combination of copeptin and high-sensitivity cardiac troponin T assay in unstable angina and non-ST-segment elevation myocardial infarc-tion: a pilot study. Arch Cardiovasc Dis. 104(1):4-10.

46. Charpentier S, Maupas-Schwalm F, Cournot M, Elbaz M, Botella J and Lauque D. (2012): Combination of copeptin and troponin assays to rapidly rule out non-ST elevation myocardial infarction in the emergency department. Acad Emerg Med. 19(5):517-24.

47. Folli C, Consonni D, Spessot M, Salvini L, Velati M, Ran-zani G, et al. (2013): Diagnostic role of copeptin in pa-tients presenting with chest pain in the emergency room. Eur J Intern Med. 24: 189-193.

48. Freynhofer M, Tajsic´ M, Wojta J and Huber K. (2012): Biomarkers in acute coronary artery disease. Wien Med Wochenschr. 162(21-22):489-498.

49. White H. (2011): Pathobiology of troponin elevations: do elevations occur with myocardial ischemia as well as ne-crosis? J Am Coll Cardiol. 57:2406-2408.

50. Vasile V and Jaffe A. (2009): The biological basis of tropo-nin in heart disease: possible uses for troponin fragmentol-ogy. Heart Metab. 43:5-8.

51. Antman E. (2012): ST-segment elevation myocardial in-farction: pathology, pathophysiology and clinical features. In: Bonow R, Mann D, Zipes D and Libby P. Braunwald’s heart disease; a textbook of cardiovascular medicine. 9th ed. Philadelphia. Saunders, an imprint of Elsevier Inc.

52. Chenvier-Gobeaux C, Freund Y, Claessens Y, Guerin S, Bonnet P, Doumenc B, et al. (2013): Copeptin for rapid rule out of acute myocardial infarction in emergency de-partment. Inter J Cardiol. 166 (1):198-204.

53. McCord J. (2008): Protocols for diagnosis of myocardial infarction. In: De Lemos J. Biomarkers in heart disease. USA. American heart association.

54. Esses D, Gallagher E, Iannaccone R, Bijur P, Srinivas V, Rose H, et al. (2001): Six-hour versus 12-hour protocols for AMI: CK-MB in conjunction with myoglobin. Am J Emer Med. 19(3):182-186.

55. Hessel M, Atsma D, van der Valk E, Bax W, Schalij M and van der Laarse A. (2008): Release of cardiac troponin I from viable cardiomyocytes is mediated by integrin stimu-

742lation. Pflugers Arch - Eur J Physiol. 455:979-986.

56. McDonough J, Arrell D and Van Eyk J. (1999): Troponin I degradation and covalent complex formation accompanies myocardial ischemia/ reperfusion injury. Circ Res. 84:9-20.

57. Feng J, Schaus B, Fallavollita J, Lee T and Canty J. (2001): Preload induces troponin I degradation independently of myocardial ischemia. Circulation. 103:2035-2037.

58. Jaffe A and Wu A. (2012): Troponin release-reversible or irreversible injury? Should we care? Clin Chem. 58(1):148-150.

59. Johnson-Davis K and McMillin G. (2010): Enzymes. In: Bishop M, Fody E and Schoeff L. Clinical chemistry; techniques, principles and correlations. 4th ed. Philadel-

phia. Lippincott Williams & Wilkins, a Wolters Kluwer publisher.

60. Sanhai W, Eloff B and Christenson R. (2010): Cardiac and muscle disease. In: Kaplan L and Pesce A. Clinical chemistry; theory, analysis and correlation. USA. Mosby, an imprint of Elsevier Inc.

61. Ishikawa Y, Saffitz J, Mealman T, Grace A and Roberts R. (1997): Reversible myocardial ischemic injury is not as-sociated with increased creatine kinase activity in plasma. Clin Chem. 43:467-475.

62. Ray P, Charpentier S, Chenevier-Gobeaux C, Reichlin T, Twerenbold R, Claessens Y, et al. (2012): Combined co-peptin and troponin to rule out myocardial infarction in patients with chest pain and history of coronary artery dis-ease. Am J Emerg Med. 30: 440-448.