Table of Contents    
ORIGINAL ARTICLE
Year : 2021  |  Volume : 12  |  Issue : 1  |  Page : 52-56  

Comparison of serum benzo(a)pyrene diol epoxide – protein adducts level between lretek cigarette smokers and nonsmokers and the related factors


1 Department of Pulmonology and Respiratory Medicine, Faculty of Medicine, Universitas Indonesia, Persahabatan Hospital, Jakarta, Indonesia
2 Department of Pulmonology and Respiratory Medicine, Persahabatan Hospital; Department of Nutrition, Faculty of Medicine, Universitas Indonesia, Dr. Cipto Mangunkusumo Hospital, Jakarta, Indonesia

Date of Submission16-May-2020
Date of Decision24-Jul-2020
Date of Acceptance01-Aug-2020
Date of Web Publication27-Jan-2021

Correspondence Address:
Agus Dwi Susanto
Department of Pulmonology and Respiratory Medicine, Faculty of Medicine, Universitas Indonesia, Persahabatan Hospital, Jalan Persahabatan Raya No. 1, Rawamangun Jakarta 13230
Indonesia
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jnsbm.JNSBM_100_20

Rights and Permissions
   Abstract 


Background: Benzopyrene is a carcinogenic agent found in cigarette smoke. Benzo(a)pyrene diol epoxide (BPDE) is one of the benzopyrene metabolites. In this study, we investigated the level of serum BPDE in kretek cigarette smokers compared to non-smokers. Methods: A cross-sectional study which involved 32 “healthy” kretek cigarette smokers and 32 “healthy” nonsmokers were conducted. We collected the blood sample and the serum BPDE level was assayed using enzyme-linked immunosorbent assay kit. The BPDE serum level in kretek cigarette smokers was compared to the level in nonsmokers. Results: A total of 32 kretek smokers and 32 controls underwent an examination of the BPDE-protein adducts level. In the kretek smokers group, 59.4% were aged over 45 years and 56.3% have a high educational background, while in the control group, 87.5% were aged under 45 years and 75% have high educational backgrounds. The level of BPDE-protein adducts in the kretek smokers subject was 12.15 (8.87–33.55) ng/ml and the levels in the control group were 11.4 (3.87–13.27) ng/ml, P = 0.004. The factors which influence the levels BDPE-protein adducts in smokers cigarettes, as determined by multivariate analysis, were sucking pattern (P = 0.002) and the degree of addiction (P = 0.047). Conclusion: The serum BPDE-protein adducts level was higher in smokers compared to nonsmokers, and the sucking pattern and degree of addiction are the influencing factors.

Keywords: Benzo(a)pyrene diol epoxide-protein adducts, nonsmokers, smokers


How to cite this article:
Susanto AD, Yusril N, Zaini J, Nuwidya F. Comparison of serum benzo(a)pyrene diol epoxide – protein adducts level between lretek cigarette smokers and nonsmokers and the related factors. J Nat Sc Biol Med 2021;12:52-6

How to cite this URL:
Susanto AD, Yusril N, Zaini J, Nuwidya F. Comparison of serum benzo(a)pyrene diol epoxide – protein adducts level between lretek cigarette smokers and nonsmokers and the related factors. J Nat Sc Biol Med [serial online] 2021 [cited 2021 Jun 14];12:52-6. Available from: http://www.jnsbm.org/text.asp?2021/12/1/52/307846




   Introduction Top


Smoking is the main cause of lung cancer in 80%–90% of lung cancer cases. Smokers are 23 times more likely to develop lung cancer than nonsmokers. Cigarettes will burn at high temperatures, produce thousands of chemicals, and cause smokers to inhale a mixture of toxic substances, and most of the substances are carcinogens and toxins.[1],[2],[3],[4],[5]

The most widely known components of cigarette smoke are tar, nicotine, and carbon monoxide. Tar contains carcinogenic polycyclic aromatic hydrocarbon (PAH) compounds. One component of PAH, benzo(a)pyrene (BaP), is known as the first chemical carcinogen found. Benzo(a)pyrene diol epoxide (BPDE) is a mutagenic active metabolite of BaP. BaP is declared as carcinogenic to humans (Group 1) in the 2010 International Agency for Research on Cancer (IARC). Cigarettes are one of the sources of high BaP levels in addition to emissions of gas and grilled food. The researchers concluded that BPDE-protein adducts in blood cells can be used as appropriate biological markers of BaP genotoxic exposure and are very promising in assessing human health risks.[1],[6],[7]

Ludovici et al. measured BPDE-protein adducts in 30 subjects divided into three groups including smokers (n = 10), former smokers (n = 5), and nonsmokers (n = 15). The level of BPDE-protein adducts in smokers was 4.46 ± 5.76 ng/ml while in nonsmokers was 1.76 ± 1.69 ng/ml.[8] An analytical study conducted by Warouw et al., who assessed the levels of BaP in highway road officers, concluded that the average inhalation intake of BaP was 3.9 μg/m3, and the estimated level of cancer risk was 11.6 new cases per 10,000 population.[9] There has been no study that related to the levels of BPDE-protein adducts in the population of kretek smokers and nonsmokers conducted in Indonesia. This study's aim was to compare the levels of BPDE-protein adducts in the population of kretek smokers and nonsmokers.


   Methods Top


A cross-sectional study was conducted at the Persahabatan Hospital, Jakarta, Indonesia, from January to March 2016. The sampling was carried out using consecutive sampling. Inclusion criteria including kretek smokers and nonsmokers, male, aged 30–60 years who were at the hospital and were willing to voluntarily participate in all research programs by signing the written informed consent. Exclusion criteria were a history of liver disease. Subjects who met the inclusion criteria were then filled out a baseline data questionnaire and Fagerstrom questionnaire underwent physical examination, and blood sampling for the BPDE examination.

The determination of BPDE-protein adducts levels was done by the enzyme-linked immunosorbent assay (ELISA) method using OxiselectTM BPDE-Protein Adduct ELISA Kit catalog number STA-301 (Cell Biolabs Inc., San Diego, CA). Samples were processed according to the manufacturer's guideline. This research has received ethical approval from the Ethics Committee of the Faculty of Medicine, Universitas Indonesia (Number: 963/UN2.F1/ETIK/2015, date 2 November, 2015).

The data obtained was then be coded, continued by data entry, and verification to be further processed statistically. The descriptive and analytic analysis was carried out. Inferential analysis to determine the differences in BPDE-protein adduct levels between kretek smokers and nonsmokers was tested with the unpaired t-test or the Mann–Whitney test if it did not meet the parametric test requirements. The Pearson correlation test was used to see the correlation between BPDE-protein adduct levels and CO exhalation and if it did not meet the parametric test requirements then the Spearman correlation test was used. Multivariate analysis was performed to the factors which influence the linear regression test. The statistical analysis was performed using the Statistical Package for Social Science (SPSS) software program version 20 (IBM Corp, Armonk, NY, USA.


   Results Top


The total subjects in this study were 64 respondents, including 32 kretek smokers subjects and 32 nonsmoker subjects. [Table 1] shows the distribution of sociodemographic characteristics of kretek smokers and nonsmokers as a comparison. Most of the age of subjects in the kretek smokers group was >45 years while in the nonsmoker group was <45 years. The education levels of respondents in both the groups were mostly highly educated. The types of occupations in the two groups were mostly working with no high risk of exposure to BaP. The neighborhoods in both groups mostly lived in environments with no high risk of BaP exposure. [Table 2] shows the characteristic distribution of subjects' smoking habits in kretek smokers. Smokers who consume cigarettes >10 cigarettes/day were more common in kretek smokers group and shallow vaping was more common in kretek smokers. Most smokers had a moderate Brinkman Index. The degree of addiction was evaluated with the Fagerstrom questionnaire and most smokers had a low degree of addiction or dependency. The last time smoking for all smokers was <8 h before sampling.
Table 1: Distribution of kretek smokers and nonsmokers subjects based on sociodemographic characteristics

Click here to view
Table 2: Distribution of kretek smoker subjects based on the smoking habits characteristic

Click here to view


The level of BPDE-protein adducts in kretek smokers and nonsmokers were determined initially. As shown in [Table 3], the levels of BPDE-protein adducts in kretek smokers were significantly higher than the levels of BPDE-protein adducts in the nonsmokers.
Table 3: Levels of benzo(a)pyrene diol epoxide protein adducts in kretek smokers and nonsmokers group

Click here to view


Next, we analyzed the BPDE-protein adducts based level on the sociodemographic characteristics. No significant differences of BPDE-protein adduct levels based on age, education level, and environment, of both kretek group and nonsmokers group, were reported [Table 4]. However, in the kretek group, subjects with a high risk of BaP exposure occupation has a statistically significant higher of BPDE-protein adducts levels as compared to those who have not high risk of BaP exposure.
Table 4: Levels of benzo(a)pyrene diol epoxide protein adducts in kretek smokers and nonsmokers group protein adducts in study subjects based on sociodemographic characteristics

Click here to view


We then determined the level of BPDE-protein adducts based on smoking habits [Table 5]. The BPDE-protein adducts levels were not statistically different in subjects based on the number of cigarettes/day, Brinkman Index, and addiction level according to the Fagerstrom questionnaire. However, subjects with deep cigarette sucking pattern have higher BPDE-protein adducts levels as compared to subjects with the vaping pattern. Furthermore, subjects who had last smoke of fewer than 8 h showed significantly higher BPDE-protein adducts levels as compared to those who had lest smoke of 8 h or more.
Table 5: The benzo(a)pyrene diol epoxide protein adducts level based on smoking habits

Click here to view


The multivariate analysis was conducted to determine the sociodemographic factors and smoking habits which influence BPDE-protein adducts levels in all kretek smoking groups by including factors with a value of <0.25 from the table of bivariate analysis test results. The multivariate analysis was carried out on factors of age, environment, occupation, sucking pattern, degree of addiction, number of cigarettes/day, and the Brinkman Index. In [Table 6], the results of multivariate tests with backward regression linear methods found that the most influential factor on BPDE-protein adducts levels in kretek smokers was a vaping pattern (P = 0.002) and addiction degree (P = 0.001).
Table 6: Factors which influence the benzo(a)pyrene diol epoxide-protein adducts levels in kretek smokers

Click here to view



   Discussion Top


In this study, we studied the level of serum BPDE in kretek cigarette smokers compared to nonsmokers. The age distribution of subjects in this study was almost the same as reported in the Indonesian Basic Health Research (Riset Kesehatan Dasar, Riskesdas).[10] Christen et al.[11] found that male kretek smokers averaged between the ages of 19–40 years. Studies by Marie et al.[12] and Ngahane et al.[13] also found that male kretek smokers were dominant at productive age.

The level of education in this study was almost the same as the study by Zhu et al.[14] which reported that the average smokers even had a moderate-high level of education but the level of education, either low education or high education, basically did not have a direct relationship with a person's smoking habits. A study by Chatterjee et al.[15] in 2011 stated that clusters or types of education have a relationship with smoking status. According to Alexopoulus et al.,[16] those who have a health-associated educational background tend not to smoke.

We found that respondents who work in an occupation with no high risk of BaP exposure were more likely to smoke. In contrast, a previous study reported that most of the patients who smoked worked in an industry with a risk of BaP exposure.[17] Furthermore, Rengrajan, et al.[18] found that respondents who worked with a high risk of BaP exposure have a habit of smoking. In our study, both the kretek smokers and nonsmokers group were mostly lived in environments with no high risk of BaP exposure. However, Wu et al.[19] stated that the high risk of diseases, including lung cancer, was because patients were living in a high-risk environment and also had a smoking habit.

In our study, the level of BPDE-protein adducts in kretek smokers was significantly higher than the levels of BPDE-protein adducts in nonsmokers. Ding et al.[20] stated that the BPDE level in kretek smokers was higher than other smokers and nonsmokers. The review conducted by Hecht et al.[21] found that the level of BPDE-protein adducts in smokers was 20–40 ng/ml and there was a significant relationship between smokers and increased levels of BPDE which cause lung cancer. Kaiserman et al.[22] found the average value of the BPDE level in kretek smoker respondents was 24.7 ng with a range of 3.36 ng to 28.39 ng.

A study by Campo et al.[23] stated that the range of BPDE-protein adducts levels in nonsmokers was 1.2-1.9 ng/m3. Neal et al.[24] explained that the average level of BPDE-protein adducts in 10 nonsmokers was lower (0.03 ± 0.01 ng/ml). Rojas et al.'s[25] study found that in 18 nonsmokers, the levels of BPDE-DNA adducts were lower than smokers and former smokers, although it was not explained the difference in average levels. In our study, BPDE-protein adducts levels of nonsmokers were slightly higher than in previous studies. Our result is different from the previous study where nonsmokers' subjects were included from rural areas which may have less air pollution.

In terms of age, our result was similar to the study by Ledesma et al.[26] who found that age was not associated with increased BPDE levels in smokers including kretek smokers. A study by Wang et al.[27] stated that a person's age was not associated with an increased toxicity effect from kretek cigarettes but an increase in toxicity levels including BaP, depending on the number of kretek cigarettes per day and the length of time that a person smokes.

The education level is still debated as a variable that affects BPDE-protein adducts levels on smokers' respondents. Koning et al.[28] stated that the higher education level increases awareness which reduces the smoking hazards. Zhang et al.[29] stated that respondents who had higher education levels were aware not to smoke to maintain quality of life.

In our study, no statistically significant relationship was found between the high-risk environment of BaP exposure and BPDE-protein adducts levels in both populations. This is probably because the subjects in our study mostly lived in environments that were not at high risk of BaP exposure. The Public Health England review explained that high-risk environments such as industrial zones, household wood burning, coal, motor vehicle exhaust emissions, and all the smoke produced from organic materials burning including cigarette smoke and in grilled foods with charcoal provide increased levels of PAH (benzopyrene).[30]

The sucking patterns of kretek smoking in this study were among the most influential factors on the BPDE level of protein adducts statistically. Our results were in line with the study of Ding et al.,[31] which stated that the pattern of sucking or behavior of patients in smoking was also one of the factors that increase the levels of BaP. They found that the smoking patterns of smokers that reach the inner volume will increase the levels of BaP and nicotine.

The results of the multivariate analysis found a significant relationship between increasing BPDE levels and nicotine dependence levels. Almost the same as the previous study who found the degree of kretek smoking addiction by 46.4% and an increase in nicotine levels would increase the level of toxicity of other ingredients.[13] Kretek cigarettes were the main source of exposure to BaP and exposure to nicotine which affects the degree of smoker dependence.[32] Therefore, reducing BaP exposure will help to reduce nicotine levels which certainly affects one's addiction.

In our study, the last time smoking <8 h had a higher mean BPDE level although it did not reach statistical threshold. Robins et al.[33] reported that environmental tobacco smoke that deposits from cigarettes in the lung (such as tar and BaP) occurred 8 h after smoking. Meanwhile, the European Food Safety Authority report stated that f BPDE levels of kretek smoke exposure were up to 10 ng/m3 for 5 h per day and BaP levels were higher from additional passive smokers around 40 ng/day.[34]


   Conclusion Top


The BPDE-protein adducts level in kretek was different between smokers and nonsmokers. The factors that influence the levels of BPDE-protein adduct in kretek smokers were vaping patterns and the addiction degree can be important for health promotion and therapy intervention. Further research can be explored to investigate the BPDE-protein adducts level as biomarkers for predictive or prognostic factor.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Samet JM, Gupta PC, Ray CS, Sauvaget C, Winn DM. Tobacco smoking and smokeless tobacco use. In: Stewart BW, Wild CP, editors. World Cancer Report 2014. France: The International Agency for Research on Cancer; 2014. p. 88-95.  Back to cited text no. 1
    
2.
World Health Organization. Cancer Fact Sheet. Geneva: World Health Organization; 2015. Available from: http://www.who.int/.mediacentre/factsheets/fs297/en. [Last accessed on 2015 Apr 05].  Back to cited text no. 2
    
3.
Forman D, Ferlay J, Stewart BW, Wild CP. The global and regional burden of cancer. In: Stewart BW, Wild CP, editors. World Cancer Report 2014. France: The International Agency for Research on Cancer; 2014. p. 16-53.  Back to cited text no. 3
    
4.
Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor A, et al. Cancer statistics, 2005. CA Cancer J Clin 2005;55:10-30.  Back to cited text no. 4
    
5.
Hoffmann D, Djordjevic MV, Hoffmann I. The changing cigarette. Prev Med 1997;26:427-34.  Back to cited text no. 5
    
6.
World Health Organization International Agency for Research on Cancer. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. France: International Agency for Research on Cancer; 2004. p. 49-263.  Back to cited text no. 6
    
7.
Hoffmann D, Hoffmann I, El-Bayoumy K. The less harmful cigarette: A controversial issue. a tribute to Ernst L. Wynder. Chem Res Toxicol 2001;14:767-90.  Back to cited text no. 7
    
8.
Lodovici M, Akpan V, Giovannini L, Migliani F, Dolara P. Benzo[a] pyrene diol-epoxide DNA adducts and levels of polycyclic aromatic hydrocarbons in autoptic samples from human lungs. Chem Biol Interact 1998;116:199-212.  Back to cited text no. 8
    
9.
Warouw SP, Kusnoputranto H, Karim F, Musadad A, Anwar A, Sukana B, et al. Analysis of benzopiren levels among highway officers [Studi analisis kadar benzopiren pada petugas jalan tol]. Dissertation. Jakarta; 2008.  Back to cited text no. 9
    
10.
Center for Health Research and Development [Badan Penelitian dan Pengembangan Kesehatan]. Basic Health Research 2010 [Riset Kesehatan Dasar 2010]. Ministry of Health, Republic of Indonesia [Kementerian Kesehatan Republik Indonesia], Jakarta, Indonesia; 2010. Available from: http://kesga.kemkes.go.id/images/pedoman/Riskesdas%202010%20Nasional.pdf. [Last Accessed on 2020 April 04].  Back to cited text no. 10
    
11.
Christen WG, Glynn RJ, Manson JE, Ajani UA, Buring JE. A prospective study of cigarette smoking and risk of age-related macular degeneration in men. JAMA 1996;276:1147-51.  Back to cited text no. 11
    
12.
Ng M, Freeman MK, Fleming TD, Robinson M, Dwyer-Lindgren L, Thomson B, et al. Smoking prevalence and cigarette consumption in 187 countries, 1980-2012. JAMA 2014;311:183-92.  Back to cited text no. 12
    
13.
Mbatchou Ngahane BH, Atangana Ekobo H, Kuaban C. Prevalence and determinants of cigarette smoking among college students: A cross-sectional study in Douala, Cameroon. Arch Public Health 2015;73:47.  Back to cited text no. 13
    
14.
Zhu BP, Giovino GA, Mowery PD, Eriksen MP. The relationship between cigarette smoking and education revisted: Implications for categorizing persons's educational status. Am J Public Health 1996;86:1582-9.  Back to cited text no. 14
    
15.
Chatterjee T, Haldar D, Malik S, Sarkar GN, Das S, LahiRI SK. A study on habits of tobacco use among medical and non-medical student of Kolkata. Lung India 2011;28:5-10.  Back to cited text no. 15
[PUBMED]  [Full text]  
16.
Alexopoulos EC, Jelastopulu E, Aronis K, Dougenis D. Cigarette smoking among university students in Greece: A comparison between medical and other students. Environ Health Prev Med 2010;15:115-20.  Back to cited text no. 16
    
17.
The WHO European Centre for Environment and Health. WHO Guidelines for Indoor Air Quality. Selected Pollutants. World Health Organization Regional Office for Europe, Bonn; 2010. Available from: https://www.euro.who.int/__data/assets/pdf_file/0009/128169/e94535.pdf. [Last Accessed on 2020 May 06].  Back to cited text no. 17
    
18.
Rengrajan T, Rajendran P, Nandakumar N, Lokeshkumar B, Rajendran P, Nishigaki L. Exposure to polycyclic aromatic hydrocarbons with special focus on cancer. Asia Pac J Trop Biomed 2015;5:182-9.  Back to cited text no. 18
    
19.
Great Lakes Commission. An assessment of Benzo(a)Pyrene air Emissions in the Great Lakes Region. International Emission Inventory Conference; 16 August, 2012. Available from: https://www3.epa.gov/ttnchie1/conference/ei20/session10/asoehl_pres.pdf. [Last Accessed on 2020 May 06].  Back to cited text no. 19
    
20.
Ding YS, Zhang L, Jain RB, Jain N, Wang RY, Ashley DL, et al. Levels of tobacco-specific nitrosamines and polycyclic aromatic hydrocarbons in mainstream smoke from different tobacco varieties. Cancer Epidemiol Biomarkers Prev 2008;17:3366-71.  Back to cited text no. 20
    
21.
Hecht SS. Tobacco smoke carcinogenesis and lung cancer. J Nation Cancer Inst 1999;91:1994-2000.  Back to cited text no. 21
    
22.
Kaisermann MJ, Rickerts WS. Carcinogenesis in tobacco smoke: Benzo(a)Pyrene from Canadian cigarette and Cigarette tobacco. Am J Public Health 1992;82:1023-6.  Back to cited text no. 22
    
23.
Campo L, Vimercati L, Carrus A, Bisceglia L, Pesatori AC, Bertazzi PA, et al. Environmental and biological monitoring of PAHs exposure in coke-oven workers at the Taranto plant compared to two groups from the general population of Apulia, Italy. Med Lav 2012;103:347-60.  Back to cited text no. 23
    
24.
Neal MS, Zhu J, Foster WG. Quantification of benzo[a] pyrene and other PAHs in the serum and follicular fluid of smokers versus non-smokers. Reprod Toxicol 2008;25:100-6.  Back to cited text no. 24
    
25.
Rojas M, Cascorbi I, Alexandrov K, Kriek E, Auburtin G, Mayer L, et al. Modulation of benzo[a] pyrene diolepoxide-DNA adduct levels in human white blood cells by CYP1A1, GSTM1 and GSTT1 polymorphism. Carcinogenesis 2000;21:35-41.  Back to cited text no. 25
    
26.
Ledesma E, Rendueles M, Díaz M. Benzo(a)pyrene penetration on a smoked meat product during smoking time. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 2014;31:1688-98.  Back to cited text no. 26
    
27.
Wang SS, Samet JM. Tobacco smoking and cancer: The promise of molecular epidemiology. Salud Publica Mex 1997;39:331-45.  Back to cited text no. 27
    
28.
Koning P, Webbink D, Martin NG. The Effect of Education on Smoking Behaviour: New Evidance from Smoking Duration of a Sample of Twins. IZA Discussion Papers 4796, Institute of Labor Economics (IZA);2010. https://ideas.repec.org/p/iza/izadps/dp4796.html. [Last Accessed on 2020 April 08].  Back to cited text no. 28
    
29.
Zhang XY, Liang J, Chen DC, Xiu MH, He J, Cheng W, et al. Cigarette smoking in male patients with chronic schizophrenia in a Chinese population: Prevalence and relationship to clinical phenotypes. PLoS One 2012;7:e30937.  Back to cited text no. 29
    
30.
Public Health England. Polycyclic Aromatic Hydrocarbons (Benzo(a)Pyrene) Toxicology Overview. PHE Centre for Radiation, Chemical and Environmental Hazards, UK; 2018. Available from: https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/737017/PAH_TO_PHE_240818.pdf. [Last Accessed on 2020 April 20].  Back to cited text no. 30
    
31.
Ding YS, Ward J, Hammond D, Watson CH. Mouth-level intake of benzo[a] pyrene from reduced nicotine cigarettes. Int J Environ Res Public Health 2014;11:11898-914.  Back to cited text no. 31
    
32.
Andrade LF, Alessi SM, Petry NM. The effects of alcohol problems and smoking on delay discounting in individuals with gambling problems. J Psychoactive Drugs 2013;45:241-8.  Back to cited text no. 32
    
33.
Robins JM, Blevins D, Shneiderman M. The effective number of cigarette inhaled daily by passive smokers: Are epidemiologic and dosinetric estimate consistent. J Hazard Mater 1990;21:215-38.  Back to cited text no. 33
    
34.
Alexander J, Benford D, Cockburn A, Cravedi JP, Dogliotti E, Domenico AD, et al. Scientific opinion of the panel on contaminants in the food chain on a request from the European commission on polycyclic aromatic hydrocarbons in food. EFSA J 2008;724:1-114.  Back to cited text no. 34
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

Top
  
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
    Abstract
   Introduction
   Methods
   Results
   Discussion
   Conclusion
    References
    Article Tables

 Article Access Statistics
    Viewed272    
    Printed2    
    Emailed0    
    PDF Downloaded49    
    Comments [Add]    

Recommend this journal