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SHORT COMMUNICATION
Year : 2012  |  Volume : 1  |  Issue : 3  |  Page : 200-203

The serum protein carbonyl content level in relation to exercise stress test


1 Department of Cardiao-Thoracic, Biomedical Research Unit in Cardiovascular Sciences (BRUCS), Faculty of Allied Health Sciences; Department of Cardiao-Thoracic Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok, Thailand
2 Department of Cardiao-Thoracic, Biomedical Research Unit in Cardiovascular Sciences (BRUCS), Faculty of Allied Health Sciences; Department of Physical Therapy, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok, Thailand
3 Department of Medicine, Buddhachinaraj Hospital, Naresuan University, Phitsanulok, Thailand
4 Department of Cardiao-Thoracic, Biomedical Research Unit in Cardiovascular Sciences (BRUCS), Faculty of Allied Health Sciences; Department of Medical Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok, Thailand

Date of Web Publication26-Dec-2012

Correspondence Address:
Sarawut Kumphune
Biomedical Research Unit in Cardiovascular Sciences (BRUCS) and Department of Medical Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok
Thailand
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Source of Support: Naresuan University, Conflict of Interest: None


DOI: 10.4103/2278-344X.105089

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  Abstract 

Background: Protein carbonyl (P) is oxidatively-modified protein with diagnostic potential for acute myocardial infarction. However, many findings indicated the elevation of serum PC content level related to exercise, which could cause false positive results and limiting the specificity for acute coronary syndrome diagnosis. This study aims to evaluate the level of serum protein carbonyl content in healthy volunteers subjected to exercise stress test (EST). Materials and Methods: Serum from healthy volunteers was collected 5-10 min before performing EST and 1 hour after the EST was achieved. The serum was collected, and the serum PC content level was determined by spectrophotometric DNPH assay. Results: The serum PC content level after exercise stress test was significantly higher than that of before performing EST (0.373 ± 0.05 nM/mg vs. 0.275 ± 0.02 nM/mg, P < 0.0001). The results demonstrated that in both male and female, serum PC content level after EST was significantly higher than that of before performing EST (0.29 ± 0.03 nM/mg vs. 0.36 ± 0.05 nM/mg P < 0.0001 in male, 0.27 ± 0.02 nM/mg vs. 0.38 ± 0.06 nM/mg P < 0.0001 in female, respectively). Conclusions: This study demonstrated that exercise stress test could result in non-specificity and false positive increasing in serum PC content level in healthy subjects, which may cause misinterpretation when using PC as cardiac marker, especially in patients, who underwent exercise stress test or patients who performing heavy physical activities.

Keywords: Acute myocardial infarction, cardiac marker, exercise stress test, oxidative stress, protein carbonyl


How to cite this article:
Mekrungruangwong T, Seenak P, Luangaram S, Thongsri T, Kumphune S. The serum protein carbonyl content level in relation to exercise stress test. Int J Health Allied Sci 2012;1:200-3

How to cite this URL:
Mekrungruangwong T, Seenak P, Luangaram S, Thongsri T, Kumphune S. The serum protein carbonyl content level in relation to exercise stress test. Int J Health Allied Sci [serial online] 2012 [cited 2019 Sep 16];1:200-3. Available from: http://www.ijhas.in/text.asp?2012/1/3/200/105089


  Introduction Top


The diagnosis of acute myocardial infarction (AMI) criteria is the history of chest pain associated with electrocardiogram) ECG) changes and biochemical markers alterations. [1] However, lack of specificity of symptom and sensitivity of ECG becomes problematic for diagnosis and delay essential intervention, especially in non-ST-segment elevation myocardial infarction (NSTEMI). [2] The early biomarkers, which reflect the early phase of cellular injury, are useful. Determination of serum protein carbonyl (PC) content is useful as a diagnostic marker for diagnosis of NSTEMI. [3] However, serum PC content level also found to be increased in exercises. [4],[5],[6],[7],[8] Therefore, we aim to examine the level of serum PC content in healthy volunteers before and after performing EST.


  Materials and Methods Top


Study population

The subject population consisted of 30 apparently healthy volunteers; 15 men and 15 women (age range 20-35 years). Subjects who had history and physical signs or symptoms of diseases such as heart disease, liver disease, renal disease, diabetes, taking antioxidant supplement, alcoholic and smoking behavior were excluded from this study. The volunteers gave written informed consent prior to the start of this study. The study protocol was approved by the Naresuan University ethical committee on human rights related to research involving human subject.

Exercise stress test

An exercise stress test (EST) performed in this study was performed using the standard Bruce protocol. [9],[10] The exercise testing began with subjects walking slowly for 3 min at 2.74 km/hr (1.7 mph) at a gradient of 10% and every 3 minutes, the speed and slope of the treadmill were progressively increased every 3 min until achieved the target heart rate (85% of maximum heart rate) or met any termination criteria such as chest or leg pain, dyspnea, blood pressure increased or decreased beyond acceptable limits. The 12-lead electrocardiogram, HR, and blood pressure were monitored during exercise. After the test was completed, the blood pressure, heart rate, and electrocardiography were collected again for further analysis.

Blood sample collection

The blood samples were collected 10 min before and 1 hr after performing exercise stress test (EST). The collected whole blood was spin down at 200 × g for 15 min. The serum was aspirated and kept under -20°C until examined. The specimen collection process was performed within 2 hours after blood collection.

Spectrophotometric determination of serum protein carbonyl

Serum protein carbonyl (PC) content was spectrophotometrically determined by colorimetric DNPH assay. Basically, protein carbonyl (PC) react with 2,4-dinitrophenylhydrazone (DNPH) producing a Schiff base that subsequently produce a corresponding hydrazone, which can be measured spectrophotometrically. [3] The serum was dilute in 1:10 with phosphate-buffered saline (PBS) to the final concentration of total protein less than 10 mg/ml. Two hundred microliter of diluted serum were mixed with 800 μl of 10 mM DNPH in 2.5 M HCl. Sample control was also prepared by adding equal volume of dilute serum to 800 μl of 2.5 M HCl. Then, the reactions were incubated at room temperature for 1 h, the dark environment with 15 min interval of vortexing. One milliliter of 20% (w/v) trichloroacetic acid (TCA) was added before incubating on ice for 5 min. After centrifugation at 10,000 × g for 10 min at 4° C, the supernatants were collected and mixed with 1 ml of 10% (w/v) TCA before spinning down at 10,000 × g for 10 min at 4°C. The protein pellet was washed for 3 times with 1 ml of 1:1 (v/v) ethanol: ethyl acetate and centrifuged at 10,000 × g for 10 min at 4° C. After final washing, the protein pellet was re-suspended in 500 μl of 6 M guanidine hydrochloride and centrifuged at 10,000 × g for 5 min at 4°C. The supernatant were collected and measured the absorbance at 370 nm using control as a blank. The protein carbonyl concentration (nmol/ml) was calculated from the equation 1. The total corrected protein amount (mg), in the final step of reaction, was calculated from a bovine serum albumin standard curve dissolved in 6 M guanidine hydrochloride and read at 280 nm. The protein amount was calculated using equation 2. Then, the carbonyl content (nmol/mg) was calculated from protein carbonyl concentration per amount of corrected protein. The within-run coefficient of variation (CV) was 5.10%, and between-day coefficient of variation (CV) was 6.84 %. The linearity regression of the test using standard albumin was 0.9691.



Statistical analysis

Data were analyzed by the GraphPad Prism version 5.00. The data were expressed as mean ± standard deviation (S.D.). The differences of PC level between before and after performing EST were analyzed by pair t test. The 95% confidential limit or P value ≤ 0.05 was considered to be statistically significant.


  Results Top


Baseline characteristics

The baseline characteristics of all healthy subjects were summarized as shown in [Table 1]. The average age of subjects was 22 years old. There was no significant different between average age of male and female subjects. In addition, there were no any differences in body weight, resting blood pressure, and resting heart rate between male and female subjects [Table 1]. The electrocardiographic data during perform exercise stress test were normal (data not shown).
Table 1: Baseline characteristic of healthy volunteers

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The elevation of serum protein carbonyl content level in healthy volunteers after exercise stress test

The serum PC content was measured in all subjects 5-10 min before performing exercise stress test (EST) and 1 hr after the test was ended. The serum was collected, and the PC content was analyzed by spectrophotometric DNPH assay. The results showed that the serum PC content level after EST was significantly higher than that of before performing EST (0.373 ± 0.05 nM/mg vs. 0.275 ± 0.02 nM/mg, P < 0.0001) [Figure 1]. The increasing in serum PC content, after performing EST, was also observed in both male and female. The results also demonstrated that in both male and female volunteers, serum PC content level after EST was significantly higher than that of before performing EST (0.29 ± 0.03 nM/mg vs. 0.36 ± 0.05 nM/mg P < 0.0001 in male, 0.27 ± 0.02 nM/mg vs. 0.38 ± 0.06 nM/mg P < 0.0001 in female, respectively) [Figure 2]a and b. However, there were no statistically different of serum PC content when compared between male and female, either before or after EST.
Figure 1: Level of serum protein carbonyl (PC) content in healthy volunteers subjected to exercise stress test

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Figure 2: (a-b) Level of serum protein carbonyl (PC) content in male and female volunteers before and after performing exercise stress test

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  Discussion Top


It has been known that proteins are concerned as a target of free radical destruction. The mechanism involved in the oxidation modification of protein is thought to occur at the monomeric level of amino acid, especially cystine, tyrosine, phenylalanine, tryptophan, histidine, and methionine. [11] The process of protein oxidation creates new functional groups such as hydroxyls and carbonyls, changing in pattern of protein folding, which is important for their activity, or binding capacity to the metal ions. [12] The outcomes of the oxidative modification of protein cause protein fragmentation, cross-linking, and unfolding, which may activate or hinder proteolytic and proteosome-mediated turnover. Ischemia-modified albumin (IMA) and protein carbonyl (PC) have been known modified proteins, which are similarly modified on the basis of oxidative stress-induced protein modification and may have diagnostic potential in acute myocardial infarction. [13],[14],[15],[16]

Determination of serum IMA was also approved by US Food and Drug Administration (FDA) in 2003 as the diagnostic test for acute myocardial infarction. [17] However, Sbarouni et al. reported the increasing in serum IMA level in patients with coronary artery disease who underwent exercise stress test (EST). [16] Although the results from this study showed the increasing of serum IMA level after performing EST, there was no interaction between IMA alteration and the results of the stress test. [16] Therefore, the determination of IMA is still useful for acute coronary syndrome diagnosis, but should be aware the interpretation of IMA result in patients who underwent EST or patients in whom the heart attack occur after performing such physical activities as it may limit the specificity for diagnosis of acute coronary syndrome and possibly cause false-positive results. Kim et al. also reported the uselessness of IMA in patients with coronary artery stenosis after performing the treadmill test. [18] The results from this study demonstrated non-significantly different of IMA level, but seem to be decreased after the treadmill test when compared to the baseline. [18]

Recently, Maneewong et.al. demonstrated the benefit of serum protein carbonyl (PC) content measurement as an early diagnostic marker for non-ST-segment elevation myocardial infarction (NSTEMI). [3] However, there were many evidences reported the increasing of serum PC content level related to exercise. [4],[5],[6],[7],[8],[19] Therefore, to ensure that the interpretation of serum PC content level is useful and not affected by the exercise stress test, we examined the level of serum protein carbonyl content in healthy volunteer after performing EST.

In this study, we showed that the performing exercise (treadmill) stress test, which is a well-established technique and is widely used to assess patients with suspected coronary heart disease, could elevate the serum PC content level in healthy volunteers. The increasing in serum PC content level, both before and after EST, was not different according to the gender. The previous study from Goldfarb et al. reported that serum PC content level found to be elevated by moderate resistance exercise. [6] So, the increasing of serum PC content level in our study may be due to the intensity of the exercise stress test protocol, which is equivalent to immediate heavy exercise.

In conclusion, our data indicated that exercise stress test could result in the increasing in serum protein carbonyl content in healthy subjects, which may cause the non-specificity and false-positive result of serum PC content level. Our study does not point out the uselessness of protein carbonyl as an early cardiac marker, but we suggest the limitation of this biomarker and aware the misinterpretation that may be occurred when the measurement of serum PC content level is performed, especially in patients who underwent exercise stress test or in patients who experience of angina after performing such physical activities.


  Acknowledgment Top


This work was supported by Naresuan University Research Endowment Fund and Faculty of Allied Health Sciences, Naresuan University.

 
  References Top

1.Johanson P, Wagner GS, Dellborg M, Krucoff MW. ST-segment monitoring in patients with acute coronary syndromes. Curr Cardiol Rep 2003;5:278-83.  Back to cited text no. 1
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2.Bassand JP, Hamm CW, Ardissino D, Boersma E, Budaj A, Fernandez-Aviles F, et al. Guidelines for the diagnosis and treatment of non-ST-segment elevation acute coronary syndromes. Eur Heart J 2007;28:1598-660.  Back to cited text no. 2
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3.Maneewong K, Mekrungruangwong T, Luangaram S, Thongsri T, Kumphune S. Combinatorial Determination of Ischemia Modified Albumin and Protein Carbonyl in the Diagnosis of NonST-Elevation Myocardial Infarction. Indian J Clin Biochem 2011;26:389-95.  Back to cited text no. 3
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4.Bloomer RJ, Fry AC, Falvo MJ, Moore CA. Protein carbonyls are acutely elevated following single set anaerobic exercise in resistance trained men. J Sci Med Sport 2007;10:411-7.  Back to cited text no. 4
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5.Fogarty MC, Hughes CM, Burke G, Brown JC, Trinick TR, Duly E, et al. Exercise-induced lipid peroxidation: Implications for deoxyribonucleic acid damage and systemic free radical generation. Environ Mol Mutagen 2011;52:35-42.  Back to cited text no. 5
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6.Goldfarb AH, Garten RS, Chee PD, Cho C, Reeves GV, Hollander DB, et al. Resistance exercise effects on blood glutathione status and plasma protein carbonyls: Influence of partial vascular occlusion. Eur J Appl Physiol 2008;104:813-9.  Back to cited text no. 6
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7.Hudson MB, Hosick PA, McCaulley GO, Schrieber L, Wrieden J, McAnulty SR, et al. The effect of resistance exercise on humoral markers of oxidative stress. Med Sci Sports Exerc 2008;40:542-8.  Back to cited text no. 7
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8.McBride JM, Kraemer WJ, Triplett-McBride T, Sebastianelli W. Effect of resistance exercise on free radical production. Med Sci Sports Exerc 1998;30:67-72.  Back to cited text no. 8
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9.Fletcher GF, Balady GJ, Amsterdam EA, Chaitman B, Eckel R, Fleg J, et al. Exercise standards for testing and training: A statement for healthcare professionals from the American Heart Association. Circulation 2001;104:1694-740.  Back to cited text no. 9
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10.Gibbons RJ, Balady GJ, Bricker JT, Chaitman BR, Fletcher GF, Froelicher VF, et al. ACC/AHA 2002 guideline update for exercise testing: Summary article: A report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee to Update the 1997 Exercise Testing Guidelines). Circulation 2002;106:1883-92.  Back to cited text no. 10
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11.Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007;39:44-84.  Back to cited text no. 11
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12.Almroth BC, Sturve J, Berglund A, Forlin L. Oxidative damage in eelpout (Zoarces viviparus), measured as protein carbonyls and TBARS, as biomarkers. Aquat Toxicol 2005;73:171-80.  Back to cited text no. 12
    
13.Dalle-Donne I, Giustarini D, Colombo R, Rossi R, Milzani A. Protein carbonylation in human diseases. Trends Mol Med 2003;9:169-76.  Back to cited text no. 13
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14.Dalle-Donne I, Rossi R, Giustarini D, Milzani A, Colombo R. Protein carbonyl groups as biomarkers of oxidative stress. Clin Chim Acta 2003;329:23-38.  Back to cited text no. 14
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15.Sbarouni E, Georgiadou P, Kremastinos DT, Voudris V. Ischemia modified albumin: Is this marker of ischemia ready for prime time use? Hellenic J Cardiol 2008;49:260-6.  Back to cited text no. 15
    
16.Sbarouni E, Georgiadou P, Panagiotakos D, Kyrzopoulos S, Tsiapras D, Voudris V, et al. Ischemia modified albumin in relation to pharmacologic stress testing in coronary artery disease. Clin Chim Acta 2008;396:58-61.  Back to cited text no. 16
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17.Van Belle E, Dallongeville J, Vicaut E, Degrandsart A, Baulac C, Montalescot G. Ischemia-modified albumin levels predict long-term outcome in patients with acute myocardial infarction. The French Nationwide OPERA study. Am Heart J 2010;159:570-6.  Back to cited text no. 17
    
18.Kim JH, Choi JH, Lee HK, Bae WH, Chun KJ, Kim YS, et al. Ischemia-modified albumin (IMA) is not useful for detecting myocardial ischemia during symptom-limited exercise stress tests. Korean J Intern Med 2008;23:121-6.  Back to cited text no. 18
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19.Revan S. Effect of acute high-intensity aerobic and anaerobic exercise on oxidative damage to lipids, proteins and DNA in untrained subjects. Afr J Pharm and Pharmaco 2011;5:6.  Back to cited text no. 19
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1]


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