Assessment of Copper Status in Patients with Paroxysmal Atrial Fibrillation

Background: Recent research has demonstrated disturbances of copper balance in the development of cardiovascular diseases. Copper status in patients with atrial fibrillation is still unknown. Serum or plasma copper and ceruloplasmin activity are widely used laboratory indicators of copper status. Purpose: То evaluate copper status in the early hours of paroxysmal atrial fibrillation and monitor its changes after sinus rhythm restoration. Methods: Serum copper levels and plasma oxidase activity of ceruloplasmin were measured in 33 patients with paroxysmal atrial fibrillation (17 males, 16 females, mean age 60.03±1.93). Indicators were measured upon hospital admission, on the 24th hour and on the 28th day after sinus rhythm restoration. Thirty-three control subjects were also enrolled in the study (17 males, 16 females, mean age 59.27±1.72) and their indicators were measured only once. Copper levels and ceruloplasmin activity were quantified by atomic absorption spectrometry and colorimetric enzymatic assay respectively. Results: Mean duration of atrial fibrillation episodes up to hospitalization was 8.64±1.03 hours (from 2 hours up to 24 hours). Upon admission, patients’ copper levels and ceruloplasmin activity were decreased compared to controls (16.77±0.59 vs. 18.30±0.45 μmol/L, p<0.05; 0.03±0.01 vs. 0.07±0.01 A530/mg protein, p<0.001 respectively). 24 hours after rhythm conversion, copper was not substantially different (17.11±0.71 vs. 18.30±0.45 μmol/L, p>0.05), whereas ceruloplasmin activity was diminished (0.05±0.01 vs. 0.07±0.01 A530/mg protein, p<0.001). On 28th day we established significant difference in neither of the indicators (17.61±0.48 vs. 18.30±0.45 μmol/L, p>0.05; 0.06±0.01 vs. 0.07±0.01 A530/mg protein, p>0.05). Conclusion: Our results provide evidence for disturbances of copper homeostasis in patients with paroxysmal atrial fibrillation. Decreased copper levels and ceruloplasmin activity during the first hours of arrhythmia and their subsequent elevation following sinus rhythm restoration give us grounds to assume that these changes are closely related to the disease pathogenesis. independent risk factor for cardiovascular incidence and mortality [11,12]. Despite the keen clinical and research interest in copper, searching in Science Direct, Scopus, PubMed and Google Scholar database done until February 2014 found single studies on copper status in patients with atrial fibrillation (AF), with ambiguous results presented [13,14]. No data were found about the copper status of such patients after sinus rhythm restoration, which would give additional grounds to seek relation with the pathogenetic mechanisms of the disease. Disturbances of copper homeostasis could be either copper deficiency or copper overload. A commonly used laboratory indicator for evaluation of copper status is the serum or plasma levels of the element itself [15]. Another indicator of copper balance in the human organism is ceruloplasmin. It is a α2-glycoprotein in its essence binding about 95% of the total circulating copper in blood. Initially the protein is synthesized as an apoprotein in the liver and subsequently, after incorporating six or seven atoms of copper in its molecule, is secreted by the hepatocytes as a holoprotein [16,17]. Three of those atoms form the so-called ‘trinuclear cluster’, responsible for the protein’s enzyme activity [18]. Initially described as a ‘copper-carrying protein’, nowadays ceruloplasmin is known to be the major multicopper oxidase in the human organism [19]. The oxidase activity of ceruloplasmin shows high sensitivity to copper levels and well characterizes copper status [17,20]. Aim To evaluate the copper status in patients with paroxysmal atrial fibrillation (PAF) (established within 48 hours) by investigating serum copper levels and plasma oxidase activity of ceruloplasmin. Mariya Negrinova Negreva1*, Svetoslav Jivkov Georgiev2, Atanas Penev Penev1, Prodanova K1, Rossitsa Borisova Georgieva3, Krasimira Prodanova5 and Albena Alexandrova Alexandrova4 1First Clinic of Cardiology, Varna University Hospital “St Marina”, Hristo Smirnenski, 1, 9000 Varna, Bulgaria 2Second Clinic of Cardiology, Varna University Hospital “St Marina”, Hristo Smirnenski, 1, 9000 Varna, Bulgaria 3National Center of Public Health and Analyses, Sofia, Bulgaria 4Laboratory of Free Radicals Processes, Institute of Neurobiology, Bulgarian Academy of Sciences, Sofia, Bulgaria 5Faculty of Applied Mathematics and Informatics, Technical University of Sofia, Bulgaria *Address for Correspondence Mariya Negrinova Negreva, MD, First Clinic of Cardiology, Varna University Hospital “St Marina”, Hristo Smirnenski, 1, 9000 Varna, Bulgaria, Tel: +359 888 487 303; Fax: +359 52 302 881; E-mail: mnegreva@abv.bg Submission: 28 April, 2014 Accepted: 06 August, 2014 Published: 11 August, 2014 Reviewed & Approved by: Dr. Hui Huang, Associate Professor, Department of Cardiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, P.R. China Research Article Open Access Journal of Cardiobiology Avens Publishing Group Inviting Innovations Avens Publishing Group Inviting Innovations

Despite the keen clinical and research interest in copper, searching in Science Direct, Scopus, PubMed and Google Scholar database done until February 2014 found single studies on copper status in patients with atrial fibrillation (AF), with ambiguous results presented [13,14].No data were found about the copper status of such patients after sinus rhythm restoration, which would give additional grounds to seek relation with the pathogenetic mechanisms of the disease.Disturbances of copper homeostasis could be either copper deficiency or copper overload.A commonly used laboratory indicator for evaluation of copper status is the serum or plasma levels of the element itself [15].
Another indicator of copper balance in the human organism is ceruloplasmin.It is a α 2 -glycoprotein in its essence binding about 95% of the total circulating copper in blood.Initially the protein is synthesized as an apoprotein in the liver and subsequently, after incorporating six or seven atoms of copper in its molecule, is secreted by the hepatocytes as a holoprotein [16,17].Three of those atoms form the so-called 'trinuclear cluster', responsible for the protein's enzyme activity [18].Initially described as a 'copper-carrying protein', nowadays ceruloplasmin is known to be the major multicopper oxidase in the human organism [19].The oxidase activity of ceruloplasmin shows high sensitivity to copper levels and well characterizes copper status [17,20].

Aim
To evaluate the copper status in patients with paroxysmal atrial fibrillation (PAF) (established within 48 hours) by investigating serum copper levels and plasma oxidase activity of ceruloplasmin.

Introduction
Copper is an essential for the human organism trace element whose functions are related to the development of connective tissue, nervous system, bones, antioxidant system etc., [1,2].Its deficit leads to bone malformations, leucopenia, anaemia etc., [3,4].

Study population
The study was conducted in the Intensive Cardiac Unit of Varna University Hospital for the period of October 2010 to May 2012.Subject to the investigation were patients with PAF (AF established within 48 hours).From 338 screened patients, consecutively were selected thirty-three (17 males, 16 females, mean age 60.03±1.93)with successful sinus rhythm restoration and lack of recurrence of the arrhythmia until the end of the study.Only patients with structurally normal hearts* were included in the study.
Thirty-three control subjects were also enrolled in the study (17 males, 16 females, mean age 59.27±1.72),so the total number of study participants were sixty-six.Controls had structurally normal hearts* and no history or electrocardiographic data for AF.The control group was formed as identical to the patient group in terms of the factors known to affect our study indicators of copper status, namely: sex, age (in decades), body mass index (BMI), bad habits (alcohol intake and smoking), accompanying diseases as well as treatment of accompanying diseases (anti-hypertensive, antidiabetic and lipidlowering drugs).
In patients, regularization of the rhythm was achieved in all patients by using the drug propafenone.It was administered following its treatment plan with total duration of maximum 24 hours [21,22].After sinus rhythm restoration, until the end of the study all patients took p.o. propafenone in a maintenance dose of 150 mg three times daily.
The onset of the AF episode was precisely determined on the basis of patient's case history, in which the beginning of arrhythmia was clearly described as a subjective feeling of "sudden onset of palpitation" continuing up to the moment of hospitalization.An electrocardiogram was performed immediately after hospitalization.The diagnosis of AF was accepted only after its confirmation by the electrocardiogram.
During the control checks electrocardiography was performed and patients were questioned for "palpitation" or similar feeling.None of the methods (anamnestic or electrocardiographic) established any recurrences of AF.
The same exclusion criteria were applied to both patients and controls (see below).
Criteria for exclusion from the study: 1. Cardiovascular diseases: ischaemic heart disease; uncontrolled hypertension; implanted device to treat rhythm conduction disorders; 2. Other diseases-renal or hepatic insufficiency; diseases of the central nervous system or endocrine system (with exclusion of type 2 diabetes, noninsulin-dependent, with good control); inflammatory and/or infectious diseases in the past three months; neoplastic, haematological or autoimmune disorders; chronic pulmonary disease; *For the purpose of this study as structurally healthy hearts were defined those, where on the basis of electrocardiography and echocardiography studies, no moderate or severe valvular disease [23], operated valvular defects, congenital heart disease, cardiomyopathies, inflammatory heart disease, systolic or diastolic heart failure, were identified.

Study protocol
Serum copper status and oxidase activity of plasma ceruloplasmin in the patients were studied in dynamics as blood samples were taken three times-immediately after admission to the ward (baseline values), twenty four hours and twenty eight days after sinus rhythm restoration.
In the control group the indicators were tested once.
To avoid diet influence on serum circulating levels of copper, patients followed their usual home diet after hospital discharge until the end of the study.During their stay in the department patients received no additional copper supply.No changes were done in the treatment of concomitant diseases including hypertension, diabetes mellitus and dyslipidemia.No additional drugs were applied to patients except for propafenone (to all patients).

Sample processing and analysis
Copper content was examined in the serum obtained from venous blood collected into serum vacutainer VACUETTE/4.0/SerumSep.Ceruloplasmin oxidase activity was determined in plasma obtained from venous blood collected into heparin vacutainer VACUETTE/4.0 ml/Li Hep.All blood samples were centrifuged at 600 g for 10 min and the separated plasma and serum were frozen at -70 ºC and preserved at this temperature for up to six months.
Serum copper levels were analysed by means of flame atomic absorption spectrometry at wavelength 324.8 nm and band pass 0.7 nm.Serum samples were diluted 1+3 with deionized water and introduced by continuous spraying into a nebulizer-burner system.The results are the average of two replicates, each with three automatic measurements; calibration is done according to a standard calibration curve [24].
Internal quality control of the copper determination was performed by analysis of certified reference material Seronorm ТМ Trace Elements Serum, Level 2, obtained from SERO AS, Norway.
The oxidase activity of ceruloplasmin was determined spectrophotometrically by the method of Ravin [25].The absorption was read at 530 nm and the enzyme activity was expressed as A 530 / mg protein.
Each plasma sample was analysed in triplicate and the obtained single results are in a good agreement (p>0.001).
All samples sent to the laboratory were coded, which did not allow the researchers to have access to data associated with the sample itself (control or patient, and the date of its collection).

Statement
All the authors of the manuscript declare that the study was performed in accordance with the Declaration of Helsinki for Human Researches [26] and after the approval of the Scientific Research Ethics Commission at the University Hospital St. Marina in Varna.All participants gave written informed consent to the study procedures.

Statistical analysis
Statistical analysis was performed using software product Graph Pad Prism 4. Descriptive statistics was used for the calculation of indicators of relative share, mean values and standard error of the mean (SEM).Analysis of the hypotheses was made through the t-criterion of Student for comparing mean values.Statistical significance was established when p<0.05.All results were presented as mean value ± SEM.

Results
The clinical characteristics of the study group and the control group are presented in Table 1.
The patients' group did not differ from controls' group in terms of number of participants in each group, age and gender structure as well as cardiovascular risk factors (p>0.05).
The statistical analysis showed that mean duration of AF episodes up to hospitalization was 8.64±1.03(from 2 hours up to 24 hours).

Discussion
It is known that despite the relatively low copper content in the human body (<100 mg), copper homeostasis is under tight control [27].It is regulated through the processes of intestinal absorption and biliary excretion [28].The liver takes central place in copper homeostasis [29].It stores the bulk of absorbed copper and controls the processes of blood secretion and biliary elimination.Owing to good regulatory mechanisms, copper levels in the body are characterized by exceptional stability [30].Therefore even with large variations in daily intake of copper from 0.8 to 7.5 mg/day, plasma levels of the trace element remain within the norm [31].
The results of our study showed that upon patients' admission to the hospital both serum levels of copper and ceruloplasmin plasma activity were decreased (Figures 1 and 2).These one-way changes in both indicators give reason to assume that there is a disturbance in the copper balance.Copper deficiency is present in the early hours (up to the 24 th hour) of the clinical manifestation of AF.It is this fact that makes it possible to assume that the established copper imbalance is most probably closely related to the mechanisms of the disease.
It has already been established that chronic AF is characterized by decreased atrial contractility and reduction in I ca amplitudes and current densities [32].Copper deficiency results in depressed peak shortening of the miocytes as well as intracellular calcium rise and decay.In this way copper deficiency may lead to impaired cardiomyocyte contractile function and calcium homeostasis [33].Influencing calcium homeostasis and cellular contractility, copper may play key role in the maintenance of effective contractility.It is quite *The smokers did not smoke more than half a packet weekly.The hospitalized patients had not smoked minimum 24-48 hours before the onset of arrhythmia.The investigations of the controls and those on the 28 th day after discharge were performed after a 48-hour period free of smoking.**No more than 1-2 drinks/weekly.The hospitalized patients had not consumed alcohol for a minimum of 48 hours before the onset of arrhythmia.The investigations of the controls and those on the 28 th day after discharge were performed after a 48-hour period without alcohol consumption possible that copper deficiency may be closely related to mechanical extension and atrial fibrillation.Further evidence is needed to clarify this relation.It would be appropriate in future studies to investigate the effect of the administration of copper products in AF recurrences.
The results obtained 24 hours after sinus rhythm restoration did not show a statistically significant difference in copper serum levels in patients and controls.Rapid increase in copper levels most probably was due to the already described strict regulatory mechanisms of copper status which restore copper balance in case of mild copper deficiency [27,30].
On the other hand, although not statistically significant, the absolute values of the 24 th hour remain in the lower borderline compared to control values (17.11±0.71 vs. 18.30±0.45μmol/L).Experimental studies have shown that lower borderline copper levels have been associated with changes in the myocardium.At sub-cellular level alterations are monitored in the number and structure of the mitochondria, and on cellular level there is significant myocyte hypertrophy with collagen deposition [15].It appears that the structure and function of the myocardium are sensitive even to marginal copper levels.These data give rise to the need for additional studies to seek the meaning of 'normal marginal values' of copper for the clinical course of AF, including its appearance and recurrence.Despite serum copper levels did not differ from those of healthy controls, ceruloplasmin activity was still significantly reduced (Figure 2).This fact could be explained by the high sensitivity of the enzyme activity to the marginal copper levels [34], while it is a prerequisite to seek connection with the other functions that the enzyme performs.
Ceruloplasmin is known to be plasma oxidase with expressed ferroxidase activity [19,35].It catalyzes the conversion of Fe 2+ into Fe 3+ which enables their binding to transferrin.That prevents the participation of Fe 2+ in the Haber-Weiss reaction and the formation of highly reactive hydroxyl radicals, which are the main initiators of lipid peroxidation.Ceruloplasmin ferroxidase activity determines it as an important antioxidant [36].The decrease in its activity is indicative of a breach in plasma antioxidant protection, which makes the issue of copper status and its implications for the pathogenesis of AF even more topical.
The results obtained 28 days after sinus rhythm restoration revealed that between patients and controls, there was no statistically significant difference in both the serum levels of copper and the enzyme activity of ceruloplasmin, which is evidence of recovery of copper balance.It is worth noting that the process occurs slowly over time.
Discussing our results, it is appropriate to note that copper status is closely related to nutritional status and that serum copper levels could be influenced by dietary copper intake.The fact raises the question whether decreased copper levels in patients with PAF were result of copper deficiency in the food.In this sense we should emphasize that during the whole period of the study none of the subjects changed his customary diet.This gives us ground to assume that changes in copper levels are unlikely to be a diet consequence.Most probably they result from impaired regulation of copper balance that may be involved in AF mechanisms.
In conclusion, we could say that the clinical manifestation of PAF is associated with changes in copper status.Copper deficiency is observed, which is transient and restores by the 28 th day after rhythm regularization.Development of copper imbalance in the early hours of atrial fibrillation (24 th hour of arrhythmia onset) is an important argument in favour of the assumption that copper is involved in the mechanisms associated with the initiation of the rhythm disorder.

3 .
Hormone replacement therapy, pregnancy, systemic administration of analgesics including NSAIDs; 4. Impossibility for the patients to clearly identify the beginning of arrhythmia.

Figure 2 :
Figure 2: Dynamics in plasma oxidase activity of ceruloplasmin in patients with PAF (A 530 /mg protein).(baseline values -values measured upon hospital admission; 24 th hour -values twenty four hours after sinus rhythm restoration; 28 th day -values measured twenty eight days after sinus rhythm restoration; **-р<0.001;ns -statistically not significant).

Table 1 :
Characteristics of the patients' and control group.