Home About us Current issue Ahead of Print Back issues Submission Instructions Advertise Contact Login   

Search Article 
  
Advanced search 
 
Saudi Journal of Kidney Diseases and Transplantation
Users online: 2370 Home Bookmark this page Print this page Email this page Small font sizeDefault font size Increase font size 
 

Table of Contents   
ORIGINAL ARTICLE  
Year : 2021  |  Volume : 32  |  Issue : 6  |  Page : 1671-1678
Study Protocol for a Randomized Controlled Trial Evaluating the Effectof Chromium Picolinate Supplementation on Gene Expression of TumorNecrosis Factor-α and DNA Damage in Metabolic Syndrome Patients


1 Nutrition and Food Security Research Center; Department of Nutrition, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
2 Department of Biostatistics and Epidemiology, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
3 Social Determinants of Health Research Center, Yasuj University of Medical Science, Yasuj, Iran
4 Cellular and Molecular Research Center, Yasuj University of Medical Sciences, Yasuj, Iran

Click here for correspondence address and email

Date of Web Publication27-Jul-2022
 

   Abstract 


Metabolic syndrome (MS) is caused by environmental factors as well as genetic. Human studies of efficacy of chromium for glucose and lipid metabolism and insulin function is not still definitive. Furthermore, the effect of chromium supplementation on the expression of inflammatory genes in patients with MS has not been studied. We will assess effects of chromium picolinate supplementation on gene expression of tumor necrosis factor-α (TNF-α) and DNA damage in MS patients. In this triple-blind randomized placebo-controlled clinical trial, 48 MS patients will be randomly assigned into two groups to receive daily 400 μg chromium picolinate supplement or placebo for 12 weeks. The outcome measures include of change in fasting blood sugar, glycosylated hemoglobin A1C, inflammatory biomarkers, lipid profile, blood pressure, gene expression of TNF-α, and 8-hydroxy-deoxyguanosine concentration as DNA damage biomarker, will be quantified at baseline and end of intervention. This protocol was approved by Institutional Research Ethics Committee School of Public Health Shahid Sadoughi University of Medical Sciences (Approval ID: IR.SSU.SPH.REC.1399.141).

How to cite this article:
Mozaffari-Khosravi H, Jambarsan S, Karimpour F, Hosseini SE, Kour BE. Study Protocol for a Randomized Controlled Trial Evaluating the Effectof Chromium Picolinate Supplementation on Gene Expression of TumorNecrosis Factor-α and DNA Damage in Metabolic Syndrome Patients. Saudi J Kidney Dis Transpl 2021;32:1671-8

How to cite this URL:
Mozaffari-Khosravi H, Jambarsan S, Karimpour F, Hosseini SE, Kour BE. Study Protocol for a Randomized Controlled Trial Evaluating the Effectof Chromium Picolinate Supplementation on Gene Expression of TumorNecrosis Factor-α and DNA Damage in Metabolic Syndrome Patients. Saudi J Kidney Dis Transpl [serial online] 2021 [cited 2022 Aug 15];32:1671-8. Available from: https://www.sjkdt.org/text.asp?2021/32/6/1671/352428



   Introduction Top


Metabolic syndrome (MS), a prevalent metabolic disorder, is defined as the existence of at least three factors of abdominal obesity, hyperglycemia, hypertension, decreased high-density lipoprotein (HDL), and hypertriglyceridemia.[1] Etiologically, obesity and resulting accumulation of fat leads to the releasing the inflammatory cytokines and ultimately insulin resistance.[2] Impaired clearance of lipid profiles from blood into tissues from insulin resistance eventually causes cardiovascular complications.[3] In addition, oxidative stress from chronic hyperglycemia can cause defective antioxidant system and exacerbation of inflammation, which leads to an increase in inflammatory biomarkers such as interleukins (ILs) and tumor necrosis factor-α (TNF-α) in blood.[4] These mechanisms leading to imbalance between antioxidant defense system and free radicals production in the body can even damage the DNA or genotoxicity[5],[6] and increased the expression of inflammatory genes.[7] This syndrome increases the risk of chronic diseases and even mortality.[7],[8],[9],[10],[11],[12] The prevalence of this syndrome has increased sharply over the past decades and has been estimated 10%–84% in different age groups in various studies.[13],[14],[15] In Iran, its prevalence was estimated to be more than 30%–50% in adults.[16],[17] In today’s machinery life, unhealthy diet (high energy intake, especially simple sugars such as fructose), sedentary lifestyle, alcohol and tobacco intake, and even lack of enough daily sleep[18] along with genetic factors[19] have been found to be effective in this pathological condition; thus, the role of nutritional interventions in prevention and reduction of its complications is important.

Chromium as an essential mineral is a cofactor for many metabolic pathways of body and is effective in improving metabolic disorders, including insulin resistance and impaired glucose metabolism. Trivalent chromium is found in fruits, vegetables, meat, fish, and brewer’s yeast.[20] Due to its low biological availability, daily requirement of chromium is not met through the usual diet.[21]

Although mechanisms of the effect of chromium on insulin resistance[22],[23] have been shown, human studies still show uncertainty and contradiction.[24] The effect of chromium supplementation on the inflammatory mediators such as IL-6 and TNF-α, although in cell and animal model studies, have been shown,[25] but in a few human studies, it has been confirmed,[26] and the exact mechanism of this effect is still unknown.[27]

To the best of our knowledge, the effect of chromium supplementation on gene expression of inflammatory biomarkers in MS has not been studied as clinical trial; therefore, the aim of this study will be to investigate the effect of trivalent chromium picolinate supplementation on gene expression of TNF-α, DNA degradation, glycemic indexes, and lipid profiles in MS patients.


   Patients and Methods Top


Study design

This study is a two-arm, triple-blind randomized placebo-controlled clinical trial. Its protocol was approved by Institutional Research Ethics Committee of University of Medical Sciences.

Sample size

Considering type, I error of 5% (a = 0.05), type II error of 20% (b = 0.20; power = 80%) and based on the following formula for calculating sample size and amount of gene expression of TNF-α as the main variable (S1, S2 = 0.25),[28] the initial number of sample in each group will be 20 persons and considering the possible loss of samples during the study, the final sample size for each group will be 24 persons.



Eligibility criteria

Patients with the following characteristics will be assigned to the study (having at least three of the five conditions):

• Waist circumference ≥88 cm for women and ≥102 cm for men

• Systolic blood pressure and diastolic greater than or equal to 130 and 85 mm Hg respectively or use of blood pressurelowering medication

• Fasting blood sugar (FBS) ≥100 mg/dL or use of hypoglycemic drugs

• Blood triglycerides (TG) ≥150 mg/dL or the use of lipid-lowering drugs

• HDL <50 mg /dL in women and less than 40 mg/dL in men[1]

• The patients will be 20–70 years old.

Exclusion criteria

The following will be excluded from the study: patients who are taking any multivitamins or dietary supplements or medications,[29] pregnant and lactating women, those who have uncontrolled blood pressure, uncontrolled blood sugar, heart arrhythmias and chronic obstructive pulmonary disease, stroke, or any other disease that requires medical treatment.[30]

Randomization

In the first stage, from registered information of people living in Dena county (SisakhatAdult Cohort Center), all eligible persons will be listed and numbered. Then, 48 patients will be randomly selected using Random Numbers Generator software. In the next step, using the above-mentioned software as a random sequencing tool, participants will be divided into two groups A (intervention) and B (placebo). Participants, laboratory technician, statistical analyst, and researcher will not be aware from patient grouping until the end of the study, and only a trusted health expert who has no involvement in the process of study will be informed. The tablets received by both groups will be the same in terms of color, smell, and appearance. The label of the boxes containing the tablets will be removed and only will be marked with a special code.

Intervention

First group (intervention) will receive 400 μg of trivalent chromium picolinate tablets (Eurho Vital company, Germany), and other groups (placebo) will receive equal amounts of placebo tablets for 12 weeks. The tablets received by both groups will be the same in terms of color, smell, and appearance. The dose of the drug was selected on the basis that despite the fact that so far no cases of poisoning and side effects of this supplement have been reported and from 100 μg to 600 μg have been prescribed in various studies,[30],[31] but a dose of 400 μg will be used. The duration of different studies had been from eight to 24 weeks,[31],[32] so to observe the relative effectiveness and prevent excessive sample loss during the study, intervention duration will be 12 weeks. An overview of the study is shown in [Figure 1]. Standard Protocol Items: Recommendations for Interventional Trials diagram including the time points of study are presented in [Table 1].
Table 1. Template of recommended content for the schedule of enrolment, interventions, and assessments.
*Weight, waist circumstance, FBS, HbA1C, blood pressure, lipid profile, IL-1, IL-6, TNF-α, 8-HDG, Gene expression of TNF-α. FBS: Fasting blood sugar, HbA1C: Hemoglobin A1C, IL: Interleukins, TNF-α: Tumor necrosis factor-α, 8-HDG: 8-hydroxy-deoxyguanosine.


Click here to view
Figure 1. Overview of the study.

Click here to view


Data collection

The primary outcomes will be gene expression related to inflammatory cytokine TNFa, Serum concentration of IL-1, 6 and TNF-α, FBS, glycosylated hemoglobin A1C (HbA1C), TG, low-density lipoprotein (LDL), HDL and total cholesterol; and the secondary outcomes: blood pressure, weigh, and waist circumference of participants.

Anthropometric measurement

Anthropometric variables will be measured both beginning and after the intervention. Participant’s weight will be quantified without shoes and with the least possible clothes with a standard scale made by Mizan Company, with an accuracy of 0.1 kg. The scales will be tested at the beginning and at time intervals when working with standard 5 kg weights. Patient’s height will be measured in standing position and without shoes, using the vertical plane of the height gauge with an accuracy of 0.1 cm. Waist circumference is also measured as the minimum value obtained between the ends of the ribs and the umbilical cord using nonstretching tape. Body mass index (BMI) will be calculated by dividing the weight [kg by the height (m2)]. Patients should be fast when measuring weight and waist circumference.

Biochemical Measurement and Gene Expression Assay

Blood sampling

At baseline and endpoint of intervention, after 12–14 overnight fasting and before taking medication, 5 mL venous blood will be taken from each patient by a laboratory technician. Blood samples are immediately centrifuged to separate serum.

Biochemical measurement

Serum concentration of FBS, glycosylated HbA1C, TG, LDL, HDL, and total cholesterol will be measured using enzymatic colorimetric method, automatic analyzer. Concentration of ILS 1, 6, and TNF-α as inflammatory markers and 8-hydroxy-deoxyguanosine (8-OH-dG) as indicator of DNA damage will be measured using enzyme-linked immunosorbent assay.

Isolation of lymphocyte cells, RNA extraction, and cDNA synthesis

From fasting blood samples, extraction of lymphocyte cells will be done using 50% precool solution (Sigma-Aldrich, Dorset, UK) gradient by centrifugation for 20 min and 3000 rpm at 4°C. Total RNA extraction will be based on acid guanidinium-phenol-chloroform procedure using RNX™-plus reagent (Cinnacolon, Tehran, Iran) according to the manufacturer’s instructions. RNAs will be treated with DNAase I (Fermentas, Lithuania) for the elimination of any genomic DNA contamination. The isolated RNA will be reverse transcribed to cDNA library using Moloney murine leukemia virus reverse transcriptase.[33]

Gene expression assay

To quantify the gene expression of TNF-α, mRNAs will be extracted using reverse transcription-polymerase chain reaction method, and primers will be designed using the Glyceraldehyde-3-phosphate dehydrogenase primers as appropriate internal standard. Graphs of gene expression will be drawn Using Light Cycler technology in the relevant software. The relative expression of the gene in the two groups will be compared with the Pffafih method or the same 2-ΔΔct method.[34]


   Statistical Analysis Top


Data were analyzed using the IBM SPSS Statistics version 23.0 (IBM Corp., Armonk, NY, USA). Continuous variables will be reported as mean ± standard deviation and categorical data as number or percentage. The normality of the distribution of variables will be checked using Kolmogorov–Smirnov test. Analyzing the differences in parametric continuous and asymmetric variables between the two groups will be done by independent samples t-test and the Mann-Whitney U test, respectively. Differences in qualitative variables between study groups will be assessed by Chi-square test. The effect of the intervention in each group will be analyzed by Paired t-test. For outcome variables with no normally distribution log transformation or two related sample tests (Wilcoxon) and Kruskal–Wallis tests will be used. For adjusting the baseline factors and individuals’ characteristics (BMI, gender, age), ANCOVA test will be applied. Statistically significant level for all tests will be considered the value P <0.05.


   Ethical consideration Top


This study protocol was registered in the Ethics Committee of University of Medical Sciences and University of Medical Sciences. The main goals and method of the study, its duration, the importance of no change in their usual habits and diet and physical activity will be described for all patients. Participation in the study will be voluntary, and leave the study during the project will be possible. Written consent will be signed by all patients. The information obtained from each subject will be kept completely confidential, and identity information of patients will be remaining secreted.


   Discussion Top


It is hypothesized that chromium supplementation as an essential element can be useful in the management of many metabolic disorders including MS, by improving blood glucose metabolism and lipid profiles, and also by reducing inflammatory mediators can improve the immune system. However, the effect of different compounds of chromium in human studies has not shown definitive results, and evidences are conflicting and may be not clinical significant[35],[36] and even without any beneficial effect;[37] so this study will be conducted to investigate the effects of chromium picolinate supplementation on glycemic index, lipid profile, blood concentration of TNF-α, and its gene expression in MS patients. The structure and design of controlled randomized selection and measurement of inflammatory gene expression along with other biochemical factors are the strengths of the study. Of course, it should be noted that although statistical tests can be used to adjust the effect of different values of diet and physical activity, because these values are based on patients’ self-report, they can be with error. It is hoped that this study can have significant effects on improving the indicators of MS.

Trial registration: Iranian Clinical Trial Registry: (IRCT20200919048757N1, at 2020- 10-18.http://www.irct.ir).

Conflict of interest: None declared.



 
   References Top

1.
Alberti KG, Eckel RH, Grundy SM, Zimmet PZ, Cleeman JI, Donato KA, et al. Harmonizing the metabolic syndrome: A joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity. Circulation 2009;120:1640- 5.  Back to cited text no. 1
    
2.
Prakash S, Rai U, Kosuru R, Tiwari V, Singh S. Amelioration of diet-induced metabolic syndrome and fatty liver with sitagliptin via regulation of adipose tissue inflammation and hepatic Adiponectin/AMPK levels in mice. Biochimie 2020;168:198-209.  Back to cited text no. 2
    
3.
Artha IM, Bhargah A, Dharmawan NK, et al. High level of individual lipid profile and lipid ratio as a predictive marker of poor glycemic control in type-2 diabetes mellitus. Vasc Health Risk Manag 2019;15:149-57.  Back to cited text no. 3
    
4.
Butkowski EG, Jelinek HF. Hyperglycaemia, oxidative stress and inflammatory markers. Redox Rep 2017;22:257-64.  Back to cited text no. 4
    
5.
Toperoff G, Aran D, Kark JD, et al. Genomewide survey reveals predisposing diabetes type 2-related DNA methylation variations in human peripheral blood. Hum Mol Genet 2012;21:371-83.  Back to cited text no. 5
    
6.
Zaki M, Basha W, El-Bassyouni HT, El-Toukhy S, Hussein T. Evaluation of DNA damage profile in obese women and its association to risk of metabolic syndrome, polycystic ovary syndrome and recurrent preeclampsia. Genes Dis 2018;5:367-73.  Back to cited text no. 6
    
7.
Aboonabi A, Aboonabi A. Anthocyanins reduce inflammation and improve glucose and lipid metabolism associated with inhibiting nuclear factor-kappaB activation and increasing PPAR-γ gene expression in metabolic syndrome subjects. Free Radic Biol Med 2020;150:30-9.  Back to cited text no. 7
    
8.
Hosseinpour-Niazi S, Tahmasebinejad Z, Esfandiar Z, Bakhshi B, Mirmiran P, Azizi F. Weight gain, but not macronutrient intake, modifies the effect of dietary branch chain amino acids on the risk of metabolic syndrome. Diabetes Res Clin Pract 2020;161: 108039.  Back to cited text no. 8
    
9.
Akbari M, Tamtaji OR, Lankarani KB, et al. The effects of resveratrol on lipid profiles and liver enzymes in patients with metabolic syndrome and related disorders: A systematic review and meta-analysis of randomized controlled trials. Lipids Health Dis.2020;19:25.  Back to cited text no. 9
    
10.
Pan Y, Kong LD. High fructose diet-induced metabolic syndrome: Pathophysiological mechanism and treatment by traditional Chinese medicine. Pharmacol Res 2018;130: 438-50.  Back to cited text no. 10
    
11.
Ashtari F, Salari M, Aminoroaya A, Deljoo BK, Moeini M. Metabolic syndrome in ischemic stroke: A case control study. J Res Med Sci 2012;17:167-70.  Back to cited text no. 11
    
12.
Wang Y, Tu R, Yuan H, et al. Associations of unhealthy lifestyles with metabolic syndrome in Chinese rural aged females. Sci Rep 2020; 10:2718.  Back to cited text no. 12
    
13.
Desroches S, Lamarche B. The evolving definitions and increasing prevalence of the metabolic syndrome. Appi Physiol Nutr Metab 2007; 32:23-32.  Back to cited text no. 13
    
14.
Kolovou GD, Anagnostopoulou KK, Salpea KD, Mikhailidis DP. The prevalence of metabolic syndrome in various populations. Am J Med Sci 2007;333:362-71.  Back to cited text no. 14
    
15.
Park SH, Farooq MA, Gaertner S, et al. Empagliflozin improved systolic blood pressure, endothelial dysfunction and heart remodeling in the metabolic syndrome ZSF1 rat. Cardiovasc Diabetol 2020;19:19.  Back to cited text no. 15
    
16.
Afkhami-Ardekani M, Zahedi-Asl S, Rashidi M, Atifah M, Hosseinpanah F, Azizi F. Incidence and trend of a metabolic syndrome phenotype among Tehranian adolescents: Findings from the Tehran Lipid and Glucose Study, 1998-2001 to 2003-2006. Diabetes Care 2010;33:2110-2.  Back to cited text no. 16
    
17.
Zabetian A, Hadaegh F, Azizi F. Prevalence of metabolic syndrome in Iranian adult population, concordance between the IDF with the ATPIII and the WHO definitions. Diabetes Res Clin Pract 2007;77:251-7.  Back to cited text no. 17
    
18.
Itani O, Kaneita Y, Tokiya M, et al. Short sleep duration, shift work, and actual days taken off work are predictive life-style risk factors for new-onset metabolic syndrome: A seven-year cohort study of 40,000 male workers. Sleep Med 2017;39:87-94.  Back to cited text no. 18
    
19.
Ichihara S, Yamada Y. Genetic factors for human obesity. Cell Mol Life Sci 2008;65: 1086-98.  Back to cited text no. 19
    
20.
Hadaegh F, Zabetian A, Tohidi M, Ghasemi A, Sheikholeslami F, Azizi F. Prevalence of metabolic syndrome by the Adult Treatment Panel III, International Diabetes Federation, and World Health Organization definitions and their association with coronary heart disease in an elderly Iranian population. Ann Acad Med Singap 2009;38:142-9.  Back to cited text no. 20
    
21.
Mahdi GS. Barley as high-chromium food. J Am Diet Assoc 1995;95:749.  Back to cited text no. 21
    
22.
Nussbaumerova B, Rosolova H, Krizek M, et al. Chromium supplementation reduces resting heart rate in patients with metabolic syndrome and impaired glucose tolerance. Biol Trace Elem Res 2018;183:192-9.  Back to cited text no. 22
    
23.
Sreekanth R, Pattabhi V, Rajan SS. Molecular basis of chromium insulin interactions. Biochem Biophys Res Commun 2008;369: 725-9.  Back to cited text no. 23
    
24.
Ngala RA, Awe MA, Nsiah P. The effects of plasma chromium on lipid profile, glucose metabolism and cardiovascular risk in type 2 diabetes mellitus. A case – Control study. PLoS One 2018;13: e0197977.  Back to cited text no. 24
    
25.
Yu Y, Tian L, Xiao Y, Huang G, Zhang M. Effect of vitamin D supplementation on some inflammatory biomarkers in type 2 diabetes mellitus subjects: A systematic review and meta-analysis of randomized controlled trials. Ann Nutr Metab 2018;73:62-73.  Back to cited text no. 25
    
26.
Imanparast F, Javaheri J, Kamankesh F, et al. The effects of chromium and vitamin D3 cosupplementation on insulin resistance and tumor necrosis factor-alpha in type 2 diabetes: A randomized placebo-controlled trial. Appl Physiol Nutr Metab 2020;45:471-7.  Back to cited text no. 26
    
27.
Jain SK, Rains JL, Croad JL. Effect of chromium niacinate and chromium picolinate supplementation on lipid peroxidation, TNF-alpha, IL-6, CRP, glycated hemoglobin, triglycerides, and cholesterol levels in blood of streptozotocin-treated diabetic rats. Free Radic Biol Med 2007;43:1124-31.  Back to cited text no. 27
    
28.
Cawood AL, Ding R, Napper FL, et al. Eicosapentaenoic acid (EPA) from highly concentrated n– 3 fatty acid ethyl esters is incorporated into advanced atherosclerotic plaques and higher plaque EPA is associated with decreased plaque inflammation and increased stability. Atherosclerosis 2010;212: 252-9.  Back to cited text no. 28
    
29.
Davari M, Hashemi R, Mirmiran P, et al. Effects of cinnamon supplementation on expression of systemic inflammation factors, NF-kB and Sirtuin-1 (SIRT1) in type 2 diabetes: A randomized, double blind, and controlled clinical trial. Nutr J 2020;19:1.  Back to cited text no. 29
    
30.
Morovati A, Pourghassem Gargari B, Sarbakhsh P. Effects of cumin (Cuminum cyminum L.) essential oil supplementation on metabolic syndrome components: A randomized, triple-blind, placebo-controlled clinical trial. Phytother Res 2019;33:3261-9.  Back to cited text no. 30
    
31.
Kim HN, Kim SH, Eun YM, Song SW. Effects of zinc, magnesium, and chromium supplementation on cardiometabolic risk in adults with metabolic syndrome: A double-blind, placebo-controlled randomized trial. J Trace Elem Med Biol 2018;48:166-71.  Back to cited text no. 31
    
32.
Derosa G, D’Angelo A, Maffioli P. Ilex paraguariensis, white mulberry and chromium picolinate in patients with pre-diabetes. Phytother Res 2020;34:1377-84.  Back to cited text no. 32
    
33.
Gmelig-Meyling F, Waldmann TA. Separation of human blood monocytes and lymphocytes on a continuous Percoll gradient. J Immunol Methods 1980;33:1-9.  Back to cited text no. 33
    
34.
Amiri Siavashani M, Zadeh Modarres S, Mirhosseini N, Aghadavod E, Salehpour S, Asemi Z. The effects of chromium supplementation on gene expression of insulin, lipid, and inflammatory markers in infertile women with polycystic ovary syndrome candidate for in vitro fertilization: A randomized, double-blinded, placebo-controlled trial. Front Endocrinol (Lausanne) 2018;9:726.  Back to cited text no. 34
    
35.
Yin RV, Phung OJ. Effect of chromium supplementation on glycated hemoglobin and fasting plasma glucose in patients with diabetes mellitus. Nutr J 2015;14:14.  Back to cited text no. 35
    
36.
Asbaghi O, Naeini F, Ashtary-Larky D, et al. Effects of chromium supplementation on lipid profile in patients with type 2 diabetes: A systematic review and dose-response metaanalysis of randomized controlled trials. J Trace Elem Med Biol 2021;66:126741.  Back to cited text no. 36
    
37.
Ghanbari M, Amini MR, Djafarian K, Shab-Bidar S. The effects of chromium supplementation on blood pressure: A systematic review and meta-analysis of randomized clinical trials. Eur J Clin Nutr 2022;76:340-9.  Back to cited text no. 37
    

Top
Correspondence Address:
Behrooz Ebrahimzadeh Kour
Nutrition and Food Security Research Center; Department of Nutrition, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd
Iran
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1319-2442.352428

Rights and Permissions


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1]



 

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


 
    Abstract
   Introduction
   Patients and Methods
   Statistical Analysis
    Ethical consider...
   Discussion
    References
    Article Figures
    Article Tables
 

 Article Access Statistics
    Viewed266    
    Printed2    
    Emailed0    
    PDF Downloaded45    
    Comments [Add]    

Recommend this journal