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Assessment of serum [epsilon]N-carboxymethyllysine and soluble receptor of advanced glycation end product levels among type 2 diabetes mellitus patients with and without acute coronary syndrome.

INTRODUCTION

In India, type 2 diabetes mellitus (T2DM) is on the way to become a pandemic. Several risk factors have being shared between T2DM and coronary artery disease (CAD) such as hyperglycemia, obesity, dyslipidemia, physical inactivity, and stress. Hence, an increased prevalence of diabetes points toward increasing risk of a CAD. [1] Diabetic patients develop CAD 2-4 times faster than the healthy population. Diabetic subjects had 21.4% prevalence of CAD, which was higher than patients with impaired glucose tolerance (14.9%) and subjects with normal glucose tolerance (9.1%). [2]

Advanced glycation end products (AGEs) are formed by non-enzymatic glycation of proteins or lipids by high serum glucose, methylglyoxal, and 3-deoxy glucosone. AGEs, mainly [epsilon]N-carboxymethyllysine (CML) has role in the development and progression of cardiovascular disease (CVD) in diabetes. [3] Hence, the serum level of AGEs was found to be high in T2DM patients with coronary heart disease (CHD) as compared to patients without CHD, and it was correlated with the severity of CHD. [4] The plaque concentrations of the specific AGEs, i.e., CML and MG-H1 were associated with inflammatory plaque markers and were higher in rupture-prone plaques (i.e., inflammatory atheromatous lesions). [5,6]

AGEs exert their effect by binding to AGE-specific cell surface receptors (RAGE) present on endothelial and smooth muscle cells. It promotes CVD in diabetes through endothelial dysfunction, inflammation, and inducing lipid abnormalities. [7,8] AGEs-RAGE binding activates endothelial cells, resulting in higher levels of endothelial adhesion molecules such as VCAM-1 and activation of transcription factor nuclear factor-kappa B, leading monocyte adhesivity and vascular permeability accelerating atherosclerosis. [4,5]

Soluble isoforms of RAGE include endogenous secretory RAGE (esRAGE) and soluble receptor of AGEs (sRAGE). Both of them lack a transmembrane domain and intracellular domain as that of RAGE, so they are devoid of intracellular signaling. Synthesis of esRAGE is by the splicing of truncated RAGE mRNA and that of sRAGE by the action of sheddase and metalloproteinase (MMP) 10 on RAGE. sRAGE has a high affinity for the ligand (AGEs) than RAGE and thus inhibits the deleterious effect of AGE-RAGE signaling. [9-11] Recombinant sRAGE blocks the development and progression of several pathological diseases such as CVS complication and neural dysfunction in animal models. [3,9] Increase in serum sRAGE levels in T2DM patients acts as a compensatory mechanism to inhibit the deleterious effect of AGE-RAGE signaling. Therefore, sRAGE was considered as antiatherogenic. [3,5,12,13]

Most studies in literature have analyzed the serum AGE and sRAGE separately so that the association between them in T2DM with acute coronary syndrome (ACS) is lacking. The exact relationship between serum AGEs and sRAGE levels among T2DM patients was not explored till date to understand the deleterious effects of AGEs and beneficial effect of sRAGE. Hence, the novelty of the study would be to collect data on serum levels of CML and sRAGE measured simultaneously among T2DM with ACS. Therefore, the objectives of the study were to measure serum CML and sRAGE levels among T2DM with and without ACS and to find out whether serum CML and sRAGE can be used to predict the risk of ACS among T2DM patients.

MATERIALS AND METHODS

Study Participants

The study was conducted in Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER) Hospital, Puducherry-06. The study was approved from Institute Research Council, and permission from Institute Human Ethics Committee was obtained (JIP/IEC/SC/2015/23/840 Dated: 21/12/2015). A written informed consent was taken from study subjects, before recruitment. 37 T2DM patients as controls were recruited based on the American Diabetes Association, 2010 criteria. [14] 37 T2DM (>18 years old) with ACS including unstable angina (UA), non-ST-elevation myocardial infarction (NSTEMI), and ST-segment elevation myocardial infarction (STEMI) were recruited based on clinical presentation, electrocardiographic changes and elevation of cardiac enzymes (Troponin-I, creatine kinase-total, and creatine kinase-MB). [14] Patients with type 1 diabetes mellitus, chronic renal disease, liver cirrhosis, congestive heart failure, chronic lung diseases, chronic infections, and smokers were excluded from the study.

Sample size Calculation

The sample size was estimated with an expected difference in the serum sRAGE level between cases and controls (mean difference of 0.20 ng/ml, with an SD of 0.17 ng/ml) at 5% level of significance and 80% power, based on the previous study. [6] It was calculated as 37 in each group.

Clinical and Biochemical Measurements

Medical history (i.e., date of birth, smoking, alcohol consumption, and medical treatments) for all subjects were recorded. Height, weight, waist circumference, and seated blood pressure were measured by the same observer. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (meter). Under the strict aseptic condition, fasting 5 mL venous blood was collected from all subjects enrolled in the study and was used for routine biochemistry investigations and CML and sRAGE analysis. Routine biochemistry investigations were assessed on fully automated analyzer by appropriate method. Serum CML and sRAGE levels were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Ray Biotech) according to the manufacturer's protocol.

Compliance with Ethical Standards

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Statistical Analysis

Categorical data were described as percentages, and continuous data were described as means [+ or -] SD. A Student's i-test (for data that were normally distributed) or a Mann-Whitney test (for data that were not normally distributed) was used to compare between cases and controls. Correlations between serum CML, sRAGE levels, and CML/sRAGE ratio and continuous variables were determined using Pearson's correlation (for normally distributed data) or Spearman's correlation (for non-normally distributed data). Receiver operating characteristic (ROC) curve was performed to investigate the value of serum CML, sRAGE levels, and CML/sRAGE ratio in differentiating T2DM patients with or without ACS. Multiple logistic regression analysis was done to look for the independent association between serum CML, sRAGE levels, and CML/sRAGE ratio and ACS adjusted for weight, waist circumference, systolic blood pressure (SBP), and BMI. A value of P < 0.05 was considered statistically significant. All analyses were performed using software package SPSS 12.0 for Windows 10.

RESULTS

Baseline Clinical Characteristics and Biochemical Measurements

Details for the (T2DM with ACS) cases and (T2DM) controls were shown in Table 1. The cases had a male predominance over the control group. Of 37 ACS patients, 7 had UA, 11 had NSTEMI, and 19 had STEMI. The cases had significantly high weight, height, waist circumference, and BMI and more alcoholics as compared to controls. There was no significant difference in blood pressure, duration of diabetes mellitus, and serum lipid profile among cases and controls.

Serum CML Concentration in Groups

Fasting serum CML concentration was higher in the cases (45.80 [+ or -] 22.25 ng/ml) than in the controls (26.04 [+ or -] 13.80 ng/ml), and the difference was statistically highly significant (P < 0.001). Patients with UA (37.24 [+ or -] 15.57 ng/ ml) had low serum CML level as compared to NSTEMI (51.04 [+ or -] 25.76 ng/ml) and STEMI (45.51 [+ or -] 22.27 ng/ml) patients [Figure 1], and the difference was statistically significant. Utilizing the ROC curve for values of CML and risk of ACS among T2DM patients, it was seen that the increase in the risk of ACS was seen at CML level more than 31.83 ng/ml (Area under curve = 0.804, sensitivity of 76%, and specificity of 78%, LR = 0.31, P < 0.0001).

Serum sRAGE Concentration in Groups

Fasting serum sRAGE concentration was higher in the cases (2.46 [+ or -] 1.43 ng/ml) than in the controls (2.16 [+ or -] 0.80 ng/ ml), but the difference was not statistically significant. No statistically difference was found in serum sRAGE among UA (1.91 [+ or -] 0.63 ng/ml), NSTEMI (2.90 [+ or -] 2.44 ng/ml), and STEMI (2.40 [+ or -] 0.64 ng/ml) patients.

Serum CML/sRAGE Ratio in Groups

Fasting serum CML/sRAGE ratio was higher in the cases (45.80 [+ or -] 22.25) than in the controls (13.17 [+ or -] 7.77), and the difference was statistically highly significant (P < 0.0001). Patients with NSTEMI (19.88 [+ or -] 6.85) had lower serum CML/ sRAGE ratio as compared to UA (20.11 [+ or -] 10.29) and STEMI (20.63 [+ or -] 12.35) patients, and the difference was statistically significant. Utilizing the ROC curve for values of CML/sRAGE ratio and risk of ACS among T2DM patients, it was seen that the increase in the risk of ACS was seen at CML/sRAGE ratio of more than 15.48 (Area under curve = 0.744, sensitivity of 70%, and specificity of 70%, LR = 0.42, P < 0.001).

Correlation between Serum CML, sRAGE Concentration, CML/sRAGE Ratio, and Clinical Parameters

Correlation analyses undertaken on all T2DM patients showed that serum CML concentration and serum CML/ sRAGE ratio were positively correlated with weight, waist circumference, BMI and serum urea, and very low-density lipoprotein cholesterol (VLDL-C). Serum sRAGE concentration did not show significant correlation with any of the physical and biochemical parameters [Table 2]. Multivariate regression (Binary logistics) analysis after adjusted for waist circumference, weight, BMI, and SBP showed that serum CML was significantly associated with ACS risk among T2DM patients [Table 3].

DISCUSSION

As the prevalence of ACS is rising, it has become necessary to find the biochemical markers that are involved in the pathogenesis of ACS. Therefore, the aim of the study was to evaluate the association between serum CML, sRAGE, and CML/sRAGE ratio with the risk of ACS in T2DM patients. In the study, there was a significant difference between cases and controls with respect to their anthropometric characters and percentage of alcoholics. There was no significant difference in the blood pressure, duration of diabetes mellitus, and serum lipid profile among cases and controls. We established that there was a high fasting serum CML concentration in cases as compared to controls which were statistically significant. Our study had demonstrated that fasting serums sRAGE concentration was low in cases compared to controls. Serum sRAGE concentration was not correlated to any physical and biochemical parameters. Correlation analyses had shown the positive association between serum CML level, CML/sRAGE ratio and weight, waist circumference, BMI and serum urea, and VLDL-C. However, serum CML level was not correlated with age, height, blood pressure, and diabetes of duration and serum total cholesterol, and triglyceride levels. Using the ROC curve, the increased risk of ACS among T2DM patients was seen at serum CML level more than 31.83 ng/ml with 76% sensitivity and 78% specificity. Although serum CML level has low sensitivity and specificity, multivariate regression (Binary logistics) analysis after adjusted for waist circumference, weight, BMI, and SBP showed that serum CML level was significantly associated with ACS risk among T2DM patients.

Falcone et al. [15] also have shown that plasma levels of sRAGE were not associated with age, duration of diabetes, smoking, duration of hypertension, BMI, creatinine, glomerular filtration rate, and C-reactive protein, but they demonstrated the inverse correlation with total cholesterol and triglycerides values. There has been an inconsistent association between serum sRAGE level and physical and biochemical parameters, raising the question of sRAGE level as a biomarker in ACS. Serum sRAGE level may not be considered as a biomarker for ACS as it is produced by proteolytic cleavage of RAGE by MMP; hence, its level depends on MMP activities and the time of blood collection. [11] Kiuchi et al. [16] also had shown no correlation between serum AGEs and age, gender, and percentage of hypertensive subjects and smoking.

This was the first kind study which showed the association between serum CML and sRAGE levels in T2DM with ACS. Increase in serum CML was one of the reasons for the occurrence of ACS in T2DM patients. Limitations of the study were as follows: (1) Diabetic patients were controls which cannot exclude confounding effects such as the effects of diseases itself and drugs treatment, (2) the different duration and different treatment of each T2DM patient might incorporate a possible source of selection bias, (3) the study population was relatively small, and (4) cause-effect relationship between serum CML levels and ACS was not possible from a cross-sectional study.

CONCLUSION

In T2DM patients with ACS, high level of serum CML and CML/sRAGE ratio was observed. The study had demonstrated that T2DM patients with high serum CML concentration and CML/sRAGE ratio were at the threat of ACS independent of other CVD risk factors and utility of measuring serum CML concentration and CML/sRAGE ratio in the prevention of future ACS in T2DM patients.

REFERENCES

[1.] Mohan V, Venkatraman JV, Pradeepa R. Epidemiology of cardiovascular disease in Type 2 diabetes: The Indian scenario. J Diabetes Sci Technol 2010;4:158-70.

[2.] Chopra S, Peter S. Screening for coronary artery disease in patients with Type 2 diabetes mellitus: An evidence-based review. Indian J Endocrinol Metab 2012;16:94-101.

[3.] Oliveira MI, de Sauza EM, Pedrosa FO, Rea RR, Alves AS, Picheth G, et al. Rage receptor and its soluble isoforms in diabetes mellitus complications. J Bras Patol Med Lab 2013;49:97-108.

[4.] Meerwaldt R, Links T, Zeebregts C, Tio R, Hillebrands JL, Smit A, et al. The clinical relevance of assessing advanced glycation endproducts accumulation in diabetes. Cardiovasc Diabetol 2008;7:29.

[5.] Piarulli F, Sartore G, Lapolla A. Glyco-oxidation and cardiovascular complications in Type 2 diabetes: A clinical update. Acta Diabetol 2013;50:101-10.

[6.] Park HJ, Baek JY, Shin WS, Kim DB, Jang SW, Shin DI, et al. Soluble receptor of advanced glycated endproducts is associated with plaque vulnerability in patients with acute myocardial infarction. Circ J 2011;75:1685-90.

[7.] Sparvero LJ, Asafu-Adjei D, Kang R, Tang D, Amin N, Im J, et al. RAGE (Receptor for advanced glycation endproducts), RAGE ligands, and their role in cancer and inflammation. J Transl Med 2009;7:17.

[8.] Ramasamy R, Yan SF, Schmidt AM. Advanced glycation endproducts: From precursors to RAGE: Round and round we go. Amino Acids 2012;42:1151-61.

[9.] Lindsey JB, Cipollone F, Abdullah SM, McGuire DK. Receptor for advanced glycation end-products (RAGE) and soluble RAGE (sRAGE): Cardiovascular implications. Diab Vasc Dis Res 2009;6:7-14.

[10.] Romero R, Espinoza J, Hassan S, Gotsch F, Kusanovic JP, Avila C, et al. Soluble receptor for advanced glycation end products (sRAGE) and endogenous secretory RAGE (esRAGE) in amniotic fluid: Modulation by infection and inflammation. J Perinat Med 2008;36:388-98.

[11.] Nakamura K, Yamagishi S, Adachi H, Matsui T, Kurita-Nakamura Y, Takeuchi M, et al. Serum levels of soluble form of receptor for advanced glycation end products (sRAGE) are positively associated with circulating AGEs and soluble form of VCAM-1 in patients with Type 2 diabetes. Microvasc Res 2008;76:52-6.

[12.] Colhoun HM, Betteridge DJ, Durrington P, Hitman G, Neil A, Livingstone S, et al. Total soluble and endogenous secretory receptor for advanced glycation end products as predictive biomarkers of coronary heart disease risk in patients with type 2 diabetes: An analysis from the CARDS trial. Diabetes 2011;60:2379-85.

[13.] Selvin E, Halushka MK, Rawlings AM, Hoogeveen RC, Ballantyne CM, Coresh J, et al. SRAGE and risk of diabetes, cardiovascular disease, and death. Diabetes 2013;62:2116-21.

[14.] Sathyaseelan AJ, Adole PS, Wyawahare M, Saya RP. Assessment of serum VASPIN levels among type 2 diabetes mellitus patients with or without acute coronary syndrome. J Clin Diagn Res 2016;10:BC07-BC10.

[15.] Falcone C, Bozzini S, D'Angelo A, Matrone B, Colonna A, Benzi A, et al. Plasma levels of soluble receptor for advanced glycation end products and coronary atherosclerosis: Possible correlation with clinical presentation. Dis Markers 2013;35:135-40.

[16.] Kiuchi K, Nejima J, Takano T, Ohta M, Hashimoto H. Increased serum concentrations of advanced glycation end products: A marker of coronary artery disease activity in type 2 diabetic patients. Heart 2001;85:87-91.

Shanija S (1), Prashant Shankarrao Adole (1), Kolar Vishwanath Vinod (2), Rama Prakasha Saya (3)

(1) Department of Biochemistry, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India, (2) Department of Medicine, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India, (3) Department of Medicine, Kanachur Institute of Medical Sciences and Research Center, Natekal, Karnataka, India

Correspondence to: Prashant Shankarrao Adole, E-mail: prashant.adole@gmail.com

Received: July 09, 2018; Accepted: July 28, 2018

DOI: 10.5455/ijmsph.2018.0721728072018

Caption: Figure 1: Construction of receiver operating characteristic curve confirmed that serum carboxymethyllysine (CML) concentration (ng/ml) and CML/soluble receptor of advanced glycation end product ratio significantly differentiated type 2 diabetes mellitus patients with or without acute coronary syndrome
Table 1: Clinical and biochemical properties of the study population

Characteristic                    Patients with T2DM

                     Without ACS (Controls)       With ACS (Cases)

Number of patients             37                        37
Age (years)           56.30 [+ or -] 13.98      54.62 [+ or -] 9.84
Male/Female                  20/17                      30/7
Weight (Kg)           67.51 [+ or -] 12.58     80.73 [+ or -] 8.96 *
Height (cm)           162.00 [+ or -] 7.35     167.11 [+ or -] 8.15 *
Waist                 86.41 [+ or -] 5.90      93.49 [+ or -] 3.70 *
  circumference
  (cm)
BMI                   25.72 [+ or -] 4.50      29.02 [+ or -] 3.85 *
Systolic blood        122.86 [+ or -] 8.39      126.48 [+ or -] 9.50
  pressure (mm of
  Hg)
Diastolic blood       81.62 [+ or -] 4.52       83.24 [+ or -] 4.75
  pressure (mm of
  Hg)
Duration of            4.89 [+ or -] 4.77        4.42 [+ or -] 3.43
  Diabetes (years)
History of                   0 (0%)                    0 (0%)
  smoking, n (%)
History of                  11 (30%)                 18 (49%) *
  alcohol, n (%)
Fasting plasma       193.62 [+ or -] 80.77     207.60 [+ or -] 64.30
  glucose (mg/dl)
Total Cholesterol    178.38 [+ or -] 45.27     175.81 [+ or -] 39.34
  (mg/dl)
Triglyceride         156.81 [+ or -] 79.04     131.08 [+ or -] 34.48
  (mg/dl)
HDL-C (mg/dl)         42.16 [+ or -] 11.25      41.40 [+ or -] 12.27
LDL-C (mg/dl)        105.05 [+ or -] 40.26     104.75 [+ or -] 36.24
VLDL-C (mg/dl)        31.32 [+ or -] 15.82      26.22 [+ or -] 7.03
CK-Total (U/l)       116.62 [+ or -] 77.35    258.64 [+ or -] 327.60 *
CK-MB (U/L)            6.47 [+ or -] 5.19      29.63 [+ or -] 30.38 *
Urea (mg/dl)          19.97 [+ or -] 5.70      26.81 [+ or -] 11.13 *
Creatinine (mg/dl)     0.89 [+ or -] 0.17       1.06 [+ or -] 0.32 *
CML (ng/ml)           26.04 [+ or -] 13.80     45.80 [+ or -] 22.25 *
sRAGE (ng/ml)          2.16 [+ or -] 0.80        2.46 [+ or -] 1.43
CML/sRAGE ratio       13.17 [+ or -] 7.77      20.31 [+ or -] 10.35 *

* P-value: < 0.05, Continuous variables were described as mean [+ or -]
SD, categorical variables were presented as frequencies. HDL-C,
high-density lipoprotein cholesterol, LDL-C: Low-density lipoprotein
cholesterol, VLDL-C: Very low-density lipoprotein cholesterol,
CK-total: Creatine Kinase-Total, T2DM: Type 2 diabetes mellitus, ACS:
Acute coronary syndrome, BMI: Body mass index, CK-MB: Creatine
kinase-MB; CML: Carboxymethyl lysine, sRAGE: Soluble Receptor of
Advance Glycation End product.

Table 2: Correlation between serum CML, sRAGE, CML/sRAGE and other
variables

Parameter                            Patients with T2 DM

                              Total (n=74)        Without ACS
                                                (Control, n=37)

                              r      P-value      r      P-value

Serum CML level
Weight (Kg)                 0.344    0.003 *    -0.014    0.389
Waist circumference (cm)    0.439    0.0001 *   0.212     0.207
(BMI)                       0.338    0.003 *    -0.020    0.907
Total Cholesterol (mg/dl)   0.052     0.658     0.397    0.015 *
LDL-C (mg/dl)               0.046     0.695     0.430    0.008 *
Urea (mg/dl)                0.262    0.024 *    -0.040    0.812
sRAGE (ng/ml)               0.449    0.0001 *   0.186     0.270
CML/sRAGE ratio             0.725    0.0001 *   0.785    0.0001 *
Serum sRAGE level
Systolic blood pressure     0.052     0.661     0.343    0.037 *
(mm of Hg)
Duration of                 0.098     0.404     0.407    0.013 *
Diabetes (years)
CML (ng/ml)                 0.449    0.0001 *   0.186     0.270
CML/sRAGE Ratio             0.725    0.0001 *   -0.394   0.016 *
Serum CML/sRAGE ratio
Weight (Kg)                 0.304    0.008 *    -0.038    0.822
Waist circumference (cm)    0.342    0.003 *    0.152     0.370
(BMI)                       0.314    0.007 *    -0.016    0.927
VLDL-C (mg/dl)              -0.245   0.035 *    -0.158    0.349
CK-MB (U/L)                 0.287    0.013 *    -0.035    0.835
CML (ng/ml)                 0.725    0.0001 *   0.785    0.0001 *
sRAGE (ng/ml)               -0.240   0.039 *    -0.394   0.016 *

Parameter                   Patients with T2 DM

                                 With ACS
                              (Cases, n=37)

                              r      P-value

Serum CML level
Weight (Kg)                 0.385     0.190
Waist circumference (cm)    0.285     0.087
(BMI)                       0.372    0.024 *
Total Cholesterol (mg/dl)   -0.147    0.386
LDL-C (mg/dl)               -0.200    0.236
Urea (mg/dl)                0.162     0.339
sRAGE (ng/ml)               0.533    0.001 *
CML/sRAGE ratio             0.624    0.0001 *
Serum sRAGE level
Systolic blood pressure     -0.127    0.453
(mm of Hg)
Duration of                 -0.089    0.600
Diabetes (years)
CML (ng/ml)                 0.533    0.001 *
CML/sRAGE Ratio             -0.283    0.090
Serum CML/sRAGE ratio
Weight (Kg)                 0.345    0.037 *
Waist circumference (cm)    0.209     0.214
(BMI)                       0.408    0.012 *
VLDL-C (mg/dl)              -0.304    0.067
CK-MB (U/L)                 0.180     0.288
CML (ng/ml)                 0.624    0.0001 *
sRAGE (ng/ml)               -0.283    0.090

* P-value: < 0.05. T2DM: Type 2 diabetes mellitus, ACS: Acute
coronary syndrome, CML: [epsilon]N-carboxymethyllysine, sRAGE:
Soluble receptor of advanced glycation end products, VLDL-C:
Very low-density lipoprotein cholesterol, CK-MB: Creatine
kinase-MB, LDL-C: Low-density lipoprotein cholesterol, BMI:
Body mass index

Table 3: Multivariate regression (Binary Logistics) analyses in all
subjects

Independent              Beta          Adjusted Odd       P-value
variables             coefficient      ratio (95%Cl)

Weight                  -0.220      0.802 (0.683-0.943)   0.007 *
Waist circumference     -0.399      0.671 (0.526-0.856)   0.001 *
BMI                      0.296      1.344 (0.850-2.125)    0.206
SBP                     -0.040      0.961 (0.879-1.051)    0.385

* P-value: < 0.05. BMI: Body mass index, CI: Confidence interval,
SBP: Systolic blood pressure
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Title Annotation:Research Article
Author:Shanija, S.; Adole, Prashant Shankarrao; Vinod, Kolar Vishwanath; Saya, Rama Prakasha
Publication:International Journal of Medical Science and Public Health
Article Type:Report
Geographic Code:9INDI
Date:Nov 1, 2018
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