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ISSN-2155-9929
Journal of Molecular Biomarkers & Diagnosis
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Serum and Saliva Levels of High-Sensitivity C-reactive Protein in Acute Myocardial Infarction

Margrét Agnarsdóttir1*, Fredrik Ponten1, Hans Garmo2,3, Gunnar Wagenius4, Lorelei Mucci5, Kristina Magnusson1, Lars Holmberg2,3 and Sonja Eaker-Fält3

1Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, SE-75185 Uppsala, Sweden

2King’s College London, Medical School, Division of Cancer Studies, SE1 9RT London, UK

3Regional Cancer Centre, Uppsala University Hospital, SE-75185 Uppsala, Sweden

4Section of Oncology, Department of Oncology and Radiology, Uppsala University, SE-75185 Uppsala, Sweden

5Department of Epidemiology, Harvard School of Public Health, 677 Huntington Avenue, Boston MA 02115, USA

*Corresponding Author:
Iraj Mirzaii-Dizgah
Department of Physiology, School of Medicine
Aja University of Medical Sciences, Tehran, Iran
Tel/Fax: +98 21 88337921
E-mail: [email protected]

Received date: June 13, 2012; Accepted date: July 11, 2012; Published date: July 13, 2012

Citation: Mirzaii-Dizgah I, Riahi E, Miri R (2012) Serum and Saliva Levels of High- Sensitivity C-reactive Protein in Acute Myocardial Infarction. J Mol Biomark Diagn 2:128. doi: 10.4172/2155-9929.1000128

Copyright: © 2012 Mirzaii-Dizgah I, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Objective: The aim of this study was to evaluate the serum and saliva levels of high-sensitivity C-reactive protein (hs-CRP) in the patients with acute myocardial infarction (MI). Materials and Methods: In a cross sectional study, 28 patients with acute MI and 28 healthy subjects were recruited to the study, and hs-CRP levels which were measured in the serum, resting saliva, and stimulated saliva at the morning of first and second days of acute MI using ELISA method. Statistical analysis of the Student’s t test and Pearson correlation coefficient were used. Results: The mean stimulated saliva hs-CRP concentration (ng/ml) was significantly higher in the patients with acute MI at both the first and the second day of MI (2.08 ± 0.55 and 2.78 ± 0.75 respectively) than in the control group (0.26 ± 0.11). It was also higher in unstimulated saliva in the patients at the both days (3.75 ± 0.92 and 2.78 ± 0.75) than controls (0.68 ± 0.21). Serum hs-CRP level (μg/ml) was higher in the patients at the second day of MI (7.03 ± 0.36) compared to healthy individuals (3.84 ± 0.60). Furthermore, stimulated and unstimulated saliva hs-CRP levels correlated significantly with serum hs-CRP level (r = 0.249, P = 0.044; r = 0.289, P = 0.038 respectively). Conclusion: It can be concluded that saliva level of hs-CRP shows a massive rise after occurrence of acute MI, and salivary hs-CRP may serve as a point-of-care testing for detection of acute MI.

Keywords

High-sensitivity C-reactive protein; Myocardial infarction; Resting saliva; Stimulated saliva

Introduction

Coronary artery disease (CAD) is the major cause of death nearly all over the world. Atherosclerosis, which is the underlying cause of most CAD, starts early in life, progresses slowly and remains subclinical for decades. Atherosclerotic process involves the interplay of inflammatory stimuli, endothelial injury and dysfunction, enhanced adhesion molecule gene expression, monocyte attraction and activation, and secretion of multiple cytokines [1-3].

Inflammation contributes to different stages in the pathogenesis of CAD. It plays a pivotal role in initiation, progression, and destabilization of atheromatous lesions, which eventually cause ischemic necrosis in acute myocardial infarction (MI). Several inflammatory parameters, such as cell adhesion molecules, cytokines, chemokines, and acute phase reactants, have been identified as valuable risk markers for the prediction of cardiovascular events. Among these, high sensitivity C-reactive protein (hs-CRP) has been central to most studies in the field. Hs-CRP predicts future coronary events merely because it is associated with all of the major risk factors for atherosclerosis, namely dyslipidemia, smoking, hypertension, diabetes, abdominal obesity, depression, other psychosocial factors and many others [2,4].

Hs-CRP plays a dual role both as a sensitive, albeit nonspecific, marker of inflammation and as an active player, increasing the risk for atherosclerosis. Its plasma concentration can rise by over 1000 fold in 24-48 hours after a strong acute stimulus such as sepsis or acute MI and can fall with a half time of about 24 h when the stimulus is removed. The main source of plasma CRP is liver. Its production appears to be regulated, during the acute phase response, by several cytokines, mainly interleukin-6, interleukin-1 and tumor necrosis factor-alpha. CRP is frequently measured in clinical practice and has been investigated extensively in the context of atherosclerosis and vascular complications [2-4].

CRP has been shown to be a prognostic marker of adverse clinical cardiac events, such as death, acute myocardial infarction, and urgent revascularization [3]. CRP measured on hospital admission for MI was associated with a strong, positive graded increase in the risk of heart failure and death independently of known prognostic factors [5].

In 2003, the American Heart Association/Centers for Disease Control recommended CRP measurement as an additional marker of prognosis in patients with acute coronary syndromes on top of characteristics in the thrombolysis in myocardial infarction risk score [4]. Hs-CRP level was found to be a stronger predictor of cardiovascular events than the LDL cholesterol level, adding prognostic information to that conveyed by the Framingham risk score [2].

In recent times there has been increasing interest in saliva-based analyses, because saliva collection methods are simple and noninvasive. Oral fluid sampling is safe for both the operator and the patient, and has easy and low-cost storage [6]. Since the saliva was put forth as a potential diagnostic tool, its use for surveillance of disease and general health has become a highly desirable goal in healthcare and medical research [7-11].

Increasing attempts to establish saliva as a diagnostic matrix have compelling reasons on behind. In this regard, it clearly offers an inexpensive, noninvasive, and easy-to-use screening method. In addition, it has several advantages over serum and urine in terms of collection, storage, shipping, and voluminous sampling. Moreover, handling of oral fluid during laboratory procedures is far easier than blood because it does not clot, thus reducing the number of required manipulations. Furthermore, the noninvasive nature of saliva collection approach could dramatically reduce anxiety and discomfort, and thereby increase patients’ willingness to continue health-related examinations over time [7].

The present study was designed to explore if saliva reflects serum concentration of hs-CRP in apparently healthy people and patients with acute ischemic coronary events. The pragmatic aim of this study was to see whether saliva can be exploited as a surrogate specimen both for diagnostic purposes in clinical practice and for screening intentions in epidemiological studies.

Methods

Study protocol and subjects

The protocol was approved by the Review Board of Aja University of Medical Sciences, and written informed consent was obtained from all patients and control subjects. The results presented in this paper were obtained from a cross sectional study which was conducted in Imam Hossein Medical Center in 2010. Thirty eight consecutive patients with typical ischemic chest pain, who were admitted to the emergency unit of Imam Hossein Medical Center, were selected to participate in the study. Based upon patients’ clinical manifestations and electrocardiographic characteristics of acute heart injury/necrosis, and/or laboratory evidence of heart-specific enzymes rises, namely creatine kinase MB (CK-MB) and cardiac troponin I, 28 (19 male and 9 female; mean age: 59 years) were recognized as patients with acute ST-elevation MI. In addition, 28 (18 male and 10 female; mean age: 60 years) age- and sex-matched individuals with no documented heart disease as control group were included in the study.

Sample collection

Venous blood and saliva were collected simultaneously from each patient with acute MI on the first and the second morning after occurrence of MI, but samples from control subjects were collected once in the morning.

For saliva sampling, all participants received detailed information about the collection protocol. The subjects swallowed all their oral fluid, and thereafter they collected 2-3 ml of their resting whole saliva in a plastic tube by spitting method with no active movement of mouth wall, sucking the oral cavity, or mastication.

Following resting saliva collection, subjects were asked to chew a piece of natural neutral gum with a given size. Two minutes after the start of chewing, subjects either spat all the oral fluid out or entirely swallowed it, and thereafter they began to collect the stimulated whole saliva into another tube while continuing chewing the gum.2 ml of venous blood were drawn immediately after saliva sampling. Upon completing sample collection, the specimens were centrifuged at 3800 g for 10 minutes, and then the serum and saliva supernatants were isolated and divided into aliquots. The aliquots were stored at -80°C for later analysis of hs-CRP.

Laboratory assays

Human CRP ELISA kits for highly sensitive measurement of CRP were provided from the company BioVendor. Determination of hs- CRP levels was carried out according to the manufacturers’ instruction. Aside from saliva samples, human sera were diluted to 1:4000 with wash buffer. The assay had a lower limit of sensitivity of 0.2ng/ml. Intra- and inter-assay coefficients of variation were less than 4.5% and 5.4%, respectively.

Statistical analysis

For statistical analysis, the data are presented as a mean ± s.e.m. Comparison of means between groups was carried out with an unpaired two-tailed student’s t-test. The Pearson correlation test was applied to determine association between serum and salivary concentration of hs-CRP. Results were considered statistically significant if P<0.05. Analyses were performed using SPSS software version 16.

Results

The mean serum concentration of hs-CRP level was higher in patients than that of controls in the second days of acute MI (Figure 1a). However, there was no significant difference in the serum hs-CRP level between groups in the first day of MI (Figure 1a).

Unstimulated salivary hs-CRP concentrations were significantly higher in patients with acute MI in both the first and the second days of acute MI (Figure 1b).

Stimulated salivary hs-CRP concentration was significantly higher in patients with acute MI, both in the first and the second days of acute MI, as compared with subjects in healthy group (Figure 1c).

molecular-biomarkers-diagnosis-high-sensitivity

Figure 1: Concentrations of high-sensitivity C-reactive protein (CRP) in serum (a) unstimulated (b) and stimulated saliva (c) of patients 12 and 24 hours after acute myocardial infarction (AMI) and control individuals.

Statistical evaluation of the data using Pearson analysis indicated a weak correlation between the unstimulated salivary concentration of hs-CRP and its serum concentration (r = 0.289, P = 0.038); and also between the stimulated salivary concentration of hs-CRP and its serum concentration (r = 0.249, P = 0.044).

Discussion

The present study has demonstrated that serum concentrations of hs-CRP are increased in the second day of acute MI. Furthermore, there is a rise of hs-CRP levels in resting whole saliva of these patients both in the first and the second days of acute MI. Moreover, stimulated saliva obtained 12 and 24 hours after onset of MI were found to have higher levels of hs-CRP.

It has been clearly established that circulating concentration of CRP is always increased after acute MI, starting 4-6 hours of onset of symptoms and reaching a peak after 50 hours [12]. There is a welldocumented interrelationship between ischemic myocardial injury and inflammatory processes. Inflammation appeared to be an important underlying mechanism of ischemic events across the spectrum of coronary syndrome, and acute MI, on the other hand, is invariably associated with acute inflammation around the ischemic lesion. Inflammatory markers such as CRP reflect the extent of myocardial necrosis and correlate with cardiac outcomes following acute MI. Moreover, myocardial necrosis following acute MI induces free radical generation and triggers the inflammatory cascade. Reperfusion therapy may also lead to further intensification of the inflammatory reaction, with the recruitment of neutrophils into the reperfused myocardium [13,14]. All of these interconnected processes may account for the massive increase of serum hs-CRP levels, a classical marker of inflammation, following an ischemic heart event.

The results of our study corroborate the rise in serum hs-CRP concentrations after MI, which in this case occurred 24 hours later. However, serum samples obtained about 12 hours after occurrence of MI displayed a substantial, albeit statistically non-significant, reduction of hs-CRP levels, a finding that is not consistent with earlier researches showing start of hs-CRP rise 4-6 hours after onset of symptoms. This observation, however, might not be simply due to elevated levels of serum hs-CRP in control subjects. The mean value of serum hs-CRP in control participants found to be 3.84 mg/l, a value that fairly falls within normal range of plasma hs-CRP concentrations, based upon recommendation made by some authors “…in routine use, a CRP value of about 3-5 mg/L, depending on the assay system, is considered normal” [15]. As a possible though not fully convincing explanation, reperfusion therapy with streptokinase might have been the reason for not elevated levels of hs-CRP in these patients. In this regard, some authors proved that in patients with acute MI who were treated with streptokinase and reperfusion was effectively achieved, CRP response was less than 20% of that in patients who did not receive thrombolytic treatment [16]. The other probable reason for this observation might be the stat treatment with ASA and atorvastatin which all patients with acute MI received as part of their routine management in the emergency unit.

In another part of our study, we demonstrated that subsequent to acute MI, hs-CRP displays substantial increases in salivary secretions of patients with MI. To the best of our knowledge, there has been just one report on detecting CRP in saliva of patients with acute MI [17], and the results of our study extend the earlier evidence and provide further details about the rise in saliva of hs-CRP. One interesting observation that emerged from this research is that salivary concentration of hs-CRP, both at resting and stimulated saliva flow rates, proved to rise earlier than serum levels, namely 12 hours following MI. As we mentioned earlier, serum hs-CRP showed reduced levels 12 hours after onset of MI, but at the same time, saliva hs-CRP displayed increased values. The reason for this observation is not clear to us. One possible explanation might be a different source of salivary hs-CRP other than circulating CRP. Apart from liver as the main source of circulating CRP, it is produced in other sites, such as respiratory tract epithelium and macrophages, kidney, neuronal cells, and even human coronary artery smooth muscle cells. Just as a pure speculation, there may be some production of CRP in oral tissues related to salivary secretions under the influence of inflammatory mediators such as interleukins 1 and 6. In this regard, it has been shown that mRNA for CRP is increased in the submandibular gland in response to experimentally induced inflammation in rats [18]. Furthermore, the ratio of mean concentration of CRP in saliva of diseased patients over that in non-MI controls was found to be far more than that of serum concentration, as shown in earlier [17], and also in our, study. This means that hs- CRP show changes that are more distinct in saliva than in serum after occurrence of MI, a finding that may favor the idea of salivary production of CRP.

Contrary to other researches that have shown serum hs-CRP remained elevated for 5-6 days after occurrence of MI [16,19], the results obtained through our survey reveals that salivary levels of hs- CRP 24 hours after onset of MI tends to be less than those of 12 hours after MI. To put it another way, salivary levels of hs-CRP begin to both elevate and then turn back to baseline prior to serum hs-CRP levels. This may support our above mentioned remark on a probably different supply of salivary hs-CRP other than circulating CRP. The moderately quick fall of saliva hs-CRP after about 24 hours of MI may be a result of flowing nature of salivary secretions, as against the relatively static quality of plasma. Circulating CRP apparently is cleared from the plasma and catabolized exclusively by hepatocytes. Its plasma half-life of about 19 hours is the same in all individuals regardless of the presence of disease or the circulating concentration of CRP. Since its plasma half-life is quite long, its rate of synthesis is the major determinant of its plasma concentration [2,4]. Nonetheless, the saliva level of hs-CRP is under the influence of saliva flow out of secretory ducts. The fairly quick fall of saliva hs-CRP may cause it to be proven useful in detecting reinfarctions.

An ideal biomarker for cardiac-related pathologies would be released early, it would ideally remain increased for some time, and it would perfectly be both sensitive and specific for the heart. CRP seemingly meets most of the criteria to be a useful indicator for cardiovascular risk assessment, and some authors have proposed that CRP can be a candidate for being used in clinical practice. Among the most important characteristics as a good biomarker, CRP is highly stable and can be stored for many years without degradation. Moreover, there is no difference in the CRP distribution curve between men and women and serum CRP levels are also independent from age and racial consideration. Thus, CRP can be measured in nonfasting individuals, with the commercially available high-sensitivity CRP assays [1,3,20,21]. Consistent with this suggestion, it has been shown that saliva-based biomarker panel of CRP, myoglobin, and myeloperoxidase in conjunction with ECG yielded strong screening capacity for detecting acute MI comparable to that of the panel brain natriuretic peptide, troponin-I, creatine kinase-MB, and myoglobin [17,22,23].

Conclusion

The results obtained through this study clearly show that subsequent to acute MI there is an enormous rise of hs-CRP levels in whole saliva at both resting and stimulated flow rates. This salivary rise and fall of hs-CRP precedes its serum counterpart. On the basis of these observations, saliva-based hs-CRP assays can be proposed as a diagnostic tool for detection of acute MI in emergency medicine.

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  1. Esmail Riahi
    Posted on Jan 30 2017 at 12:05 pm
    I'm writing to claim as an author of the article entitled "Serum and Saliva Levels of High-Sensitivity C-reactive Protein in Acute Myocardial Infarction". Unfortunately, the list of authors has been appeared erroneously in the title page. Additionally, the pdf file associated with the article is not the correct one. Please kindly double-check and correct the author list and the pdf version of the paper. Bests, Esmail riahi
 

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