Comparison of 99mTc-N-DBODC5 and 99mTc-MIBI of Myocardial Perfusion Imaging for Diagnosis of Coronary Artery Disease

Despite recent advances in therapeutic and diagnostic approaches, coronary artery disease (CAD) and its related cardiac disorders represent the most common cause of death in the United States. Nuclear myocardial perfusion imaging (MPI) technologies play a pivotal role in the diagnosis and treatment design for CAD. Recently, in order to develop improved MPI agents for diagnosis of CAD, 99mTc-[bis(dimethoxypropylphosphinoethyl)-ethoxyethyl-amine(PNP5)]-[bis(N-ethoxyethyl)dithiocarbamato(DBODC)]nitride(N-DBODC5)(99mTc-N-DBODC5) with a faster liver clearance than conventional single-photon emission computed tomography (SPECT) imaging agents (technetium 99m sestamibi (99mTc-MIBI) or technetium 99m tetrofosmin) has been introduced. In preclinical and phase I studies, 99mTc-N-DBODC5 has shown characteristics of an essentially ideal MPI tracer. Importantly, however, there is no data to support the use of 99mTc-N-DBODC5 to evaluate myocardial ischemia in patients with suspected CAD. The present study was designed to assess the clinical value of this agent; the findings of stress and rest MPI after the administration of this agent were compared to those of stress and rest 99mTc-MIBI, as well as those of coronary angiography, with respect to the detection of CAD. Our findings indicated the usefulness of 99mTc-N-DBODC5 as a promising MPI agent.


Introduction
Coronary artery disease (CAD) remains the single greatest cause of death in men and women in the USA, despite a declining total death rate. Using 2005 data, over 445,000 (or 1 in every 5) deaths in the USA were due to CAD, and it ranked highest among all disease categories in hospital discharges [1]. CAD also remains the third leading cause of death of the Chinese population, creating a significant socioeconomic burden [2]. Therefore, the reduction of the morbidity and mortality due to CAD is of primary importance to physicians and patients.
Nuclear cardiology, cardiovascular magnetic resonance, cardiac computed tomography, position emission computed tomography, and coronary angiography (CA) are imaging modalities that have been used to measure myocardial perfusion, left ventricular function, and coronary anatomy for clinical management and research [3]. Based on current guidelines, invasive CA is a suitable diagnostic procedure for patients with a high pretest likelihood of significant coronary artery disease either with or without troublesome symptoms or clinical findings [4,5]. In this population, the reported diagnostic yield of invasive CA is 44-48% [6][7][8]. However, in reality, invasive CA has an even lower diagnostic yield of obstructive CAD of approximately 38% [9]. Noninvasive testing could be of value to defer from invasive diagnostic procedures. Stress myocardial perfusion imaging (MPI) has emerged as an important noninvasive mean of evaluating patients with suspected CAD, with over 8.5 million evaluations performed annually in the USA [10].
The most commonly used imaging modality for this purpose is single-photon emission computed tomography (SPECT) [11]. The advantages of nuclear MPI for the detection of CAD are as follows: first, it enables the simple, safe, and noninvasive assessment of myocardial ischemia and reduction of coronary flow reserve using exercise or pharmacological stress. In addition, it provides left ventricular functional information on coronary arteries, which is different from the morphological information provided by CA [12]. Furthermore, CA is the standard technique for assessing epicardial coronary anatomy, and MPI is the standard technique for assessing myocardial perfusion and is appropriate for the quantitative evaluation of myocardial perfusion conditions [13].
Thallium-201 ( 201 Tl), technetium 99m sestamibi ( 99m Tc-MIBI), and technetium 99m tetrofosmin ( 99m Tc-tetrofosmin) are three traditional, routinely used, MPI tracers and are clinical-validated tracers for evaluation of SPECT MPI [14][15][16]. Because 201 Tl is a cyclotron-produced isotope, it is expensive, and it is not easily available for routine clinical use in many developing countries. Therefore, from a practical standpoint, 99m Tc-MIBI or 99m Tc-tetrofosmin is the preferred agent for a gated SPECT study. However, tracer activity below the diaphragm is commonly seen with 99m Tc-MIBI and 99m Tc-tetrofosmin, and this can reduce accuracy in some studies [17,18].
Recently, 99m Tc-N-DBODC5 with a faster liver clearance than conventional SPECT imaging agents has been introduced. It is a new lipophilic, monocationic 99m Tc-labeled compound that is currently under clinical investigation as a MPI agent [19][20][21][22][23][24]. Basic research and phase I studies have shown the safety, excellent biodistribution, and high image quality of this radiopharmaceutical [22,25,26]. Like 99m Tc-MIBI and 99m Tc-tetrofosmin, 99m Tc-N-DBODC5, also a monocationic lipophilic complex, is possibly localized in the mitochondrial fraction. Marmion et al. [27] and Bolzati et al. [28] reported that the lipophilic characteristics and electronic charge of the tracer were thought to be important. Importantly, however, the clinical value of diagnosis of CAD has not been fully explored.
In order to evaluate the clinical value of this agent, the findings of stress and rest MPI after the administration of this agent were compared to those of stress and rest 99m Tc-MIBI MPI, as well as those of CA, with respect to the detection of CAD.

Patient Population.
Of 120 patients admitted to the hospital because of chest pain from March 2010 to December 2011, 46 (31 males; mean age, 60.08 ± 8.58 years (Table 1)) who underwent stress-rest 99m Tc-N-DBODC5 SPECT, 99m Tc-MIBI SPECT, and coronary arteriography were included in this study. If the creatine kinase concentration was more than twice the upper normal limit or there was positive troponin T, or if there was evidence of previous myocardial infarction, coronary angioplasty, or a coronary artery bypass graft surgery, the patients were excluded.

Preparation of 99
Tc-N-DBODC5. 99m Tc-N-DBODC5 was prepared using a lyophilized kit formulation purchased from Beijing Shihong Pharmaceutical Center. The saline solution of sodium pertechnetate (1.0 mL, 1110 MBq) was added to an SDH vial (Vail A: 5.0 mg of succinate dehydrogenase, 5.0 mg of 1,2-diaminopropane-N,N,N ,N -tetraacetic acid, and 0.05 mg of SnCl 2 ⋅2H 2 O). Then, the solution was Radiochemical purity (RCP) was determined by thinlayer chromatography (TLC). TLC was conducted on polyamide film as the station phase and saline/acetone (v / v = 6 : 1) as the mobile phase. Retention factor (Rf) for 99m Tc-N-DBODC5 is 0.3-0.6. The RCP was more than 95% in each experimental study, and the purity was still greater than 95% at least 6 h. Figure 1 shows that a two-day protocol was used for both 99m Tc-MIBI and 99m Tc-N-DBODC5 MPI. Image acquisition procedures conformed to the ASNC imaging guidelines [29,30], and the aged-adjusted maximal heart rate was the endpoint on an ergometer, but other endpoints included physical exhaustion, uncommon arrhythmia, severe angina, or significant hypotension. At the endpoint of the symptom-limited bicycle ergometer exercise, 740 MBq of 99m Tc-N-DBODC5 (mean injected activity, 740.8 ± 11.1 MBq) was injected; exercise was continued for a further 2 min.

Exercise Protocol.
Each patient underwent an exercise 99m Tc-MIBI study within 7 days of the 99m Tc-N-DBODC5 study. For the 99m Tc-MIBI (741.5 ± 12.3 MBq) SPECT study, the exercise protocol of patients was identical to that for the 99m Tc-N-DBODC5 study. 99m Tc-N-DBODC5 and 99m Tc-MIBI were used as myocardial perfusion agents in a random sequence.

Gated SPECT Acquisition and Image Reconstruction.
Patients were imaged in the supine position with their arms raised. SPECT images were acquired with a fixed 90 ∘ twoheaded gamma camera (Infinia VC Hawkeye, General Electric, USA), using a low-energy, high-resolution, parallel-hole collimator, from the 45 ∘ right anterior oblique to the 45 ∘ left posterior position. Acquisition parameters were as follows: detectors at 90 ∘ , 180 ∘ rotation at 3 ∘ steps with automatic body contouring, 35 s acquisition per step, 64 × 64 matrix, zoom ×1.28, and energy window 140 keV ± 10%. The total acquisition time was approximately 20 min. The same filters were used for both tracers. The raw projection datasets were filtered with a Butterworth filter (cut off frequency 0.50 cycles/pixel and power 6.0 for rest images, cut off frequency 0.50 cycles/pixel and power 5.0 for stress images). No scatter or attenuation correction was performed.

Heart-to-Organ Analysis. 99m
Tc-N-DBODC5 and 99m Tc-MIBI heart-to-organ count ratios were calculated from the anterior projection of each tomographic acquisition. Regions of interests (ROIs) were drawn around the entire left ventricular myocardium, over the hepatic margin adjacent to the inferoapical wall of the left ventricle, excluding the biliary tree, and over the inferior left ventricular wall adjacent large intestine activity. The mean counts per pixel in the three ROIs were normalized to the injected tracer activity after decay correction and to a standard acquisition time of 1 min. Heartto-liver and heart-to-intestine ratios were then computed [31].

Image Quality
Assessing. For MPI analyses, at first, two experienced observers judged which image sets from 99m Tc-MIBI and 99m Tc-N-DBODC5 were superior in image quality on the basis of patient motion, statistical noise, tracer activity below the diaphragm, heart-to-organ count ratio, and sharpness, without knowledge of the radiopharmaceutical or patient identity [32].

MPI Image Interpretation.
Both overall qualitative diagnosis and semiquantitative 17 segment with 5-point [33] (0 = normal, 4 = absent tracer uptake) scorings were employed in the independent, blinded read by two expert readers who were experienced in SPECT MPI interpretation. In this model, the left anterior descending artery (LAD) distribution territory comprises seven segments (segments 1-2, 7-8, 13-14, and 17), the left circumflex artery (LCX) comprises five segments (segments 3-4, 9-10, and 15), and the right coronary artery (RCA) comprises five segments (segments 5-6, 11-12, and 16). SPECT stress/rest studies were classified for each myocardial region in the following manner: normal if all the segments were normal after stress; ischemic if at least one segments improved at rest; and scar if no segments improved at rest. In addition, the summed stress (SSS), summed rest (SRS), and summed difference scores (SDSs) were calculated, and ischemia was defined as SDS ≥ 2 [34]. The 99m Tc-MIBI and 99m Tc-N-DBODC5 images were separately interpreted without knowing the clinical histories, results of coronary angiography, or other radionuclide findings.

Coronary Angiography Interpretation.
All coronary angiograms were interpreted with quantitative CA in a coronary angiography core laboratory blinded to the clinical or imaging results. A coronary stenosis was considered present when there was a stenosis ≥ 50% in diameter in any epicardial coronary artery. The presence of one or more coronary stenoses defined the presence of significant CAD.

Statistical Analysis.
All statistical analyses were performed using the statistical package SPSS 16.0 (Chicago, IL, USA). The results were expressed as the mean value ± SD. The difference in left ventricular function parameters and perfusion scores were compared using a paired Student's t-test. Comparison of the proportion was made with the McNemar test. CA results were used as the "gold standard. " Agreements between CA and SPECT results were defined as the kappa ( ) value. < 0.05 was considered significant.

Coronary Angiography.
Of the 46 patients studied, 29 had ≥50% luminal diameter stenosis in at least one major coronary vessel. Fourteen had single-vessel disease, 11 had two-vessel disease, and four had three-vessel disease; 15 patients showed significant stenosis in the RCA, 19 in the LAD, and 14 in the LCX. The remaining 17 patients had normal or nonsignificantly stenosed coronary arteries. Heart-to-intestine ratio * * * P < 0.05 versus 99m Tc-MIBI (b) Figure 3: Heart-to-liver ratio and heart-to-intestine ratio measured with an anterior projection images at stress for 30 min and rest for 60 min for 99m Tc-N-DBODC5 and 99m Tc-MIBI in 46 patients.

3.2.
Heart-to-Organ Count Ratio. Qualitative analyses of images acquired of 99m Tc-N-DBODC5 both at rest and during stress revealed no significant overlap between tracer accumulation in the inferior wall of the ventricle and in the subdiaphragmatic region ( Figure 2). Figure 3 depicts the heart-to-liver count ratios (a) and heart-to-intestine count ratios (b) of 99m Tc-N-DBODC5 and 99m Tc-MIBI in 46 patients. The myocardium-to-liver ratios of 99m Tc-N-DBODC5 and 99m Tc-MIBI studies were 0.97 ± 0.07 and 0.51 ± 0.02, respectively, at stress. The myocardium-toliver ratios of 99m Tc-N-DBODC5 and 99m Tc-MIBI MPI were 0.78 ± 0.04 and 0.62 ± 0.01, respectively, at rest. On the other hand, the mean heart-to-intestine ratios of 99m Tc-N-DBODC5 were 2.12 ± 0.01 and 1.36 ± 0.02 for stress and rest images, respectively. The average heart-to-intestine ratios of 99m Tc-MIBI were 1.62 ± 0.01 and 0.91 ± 0.00 for stress and rest images, respectively. 99m Tc-N-DBODC5 studies had a significantly higher heart-to-organ count ratio compared with 99m Tc-MIBI studies ( < 0.05).

Image Quality.
In the stress perfusion imaging, 99m Tc-N-DBODC5 MPI was superior in quality to 99m Tc-MIBI MPI in 37 of the 46 patients studied. In the remaining nine patients, the MPI was judged to be of equal quality. In the resting perfusion imaging, 99m Tc-N-DBODC5 images were superior in all the patients studied. In general, the 99m Tc-N-DBODC5 MPI had more counts with less statistical noise, less tracer activity below the diaphragm, and desirable heart-to-organ count ratio compared with 99m Tc-MIBI MPI. . Seventy-one segments interpreted as normal on 99m Tc-N-DBODC5 corresponded to nonreversible defects on 99m Tc-MIBI SPECT images. In contrast, 12 normal 99m Tc-MIBI segments corresponded to nonreversible defects on 99m Tc-N-DBODC5 images. When the patterns of uptake (normal, reversible defect, and nonreversible defect) of 99m Tc-N-DBODC5 and 99m Tc-MIBI were compared, there was concordance in 71% (557/782) of segments. Table 3 shows the results of left ventricular function parameters in the two techniques used. In this study, the end-diastolic volume (EDV), end-systolic volume (ESV), left ventricular ejection fraction (LVEF), and transient ventricular dysfunction (TID) were assessed by MPI using two tracers. No statistically significant differences in MPI parameters were observed between the 99m Tc-MIBI and 99m Tc-N-DBODC5 studies. However, the scores of myocardial perfusion defects for the detection of CAD were higher with 99m Tc-MIBI than with 99m Tc-N-DBODC5 ( = 0.012, for SSS; = 0.020, for SDS, resp.). Table 4 shows the sensitivity and specificity of two tracers in diagnosing CAD. Based on CA,  overall figures for sensitivity and specificity in identification of CAD were 86% (25/29) and 65% (11/17) for 99m Tc-MIBI imaging. On the other hand, the overall sensitivity and specificity for the detection of CAD were 86% (25/29) and 88% (15/17) for 99m Tc-N-DBODC5 SPECT studies. The concordance of ischemia diagnosis sensitivity of a representative patient for the two tracers is shown in Figure 4. The accuracy was 0.78 for 99m Tc-MIBI and 0.87 for 99m Tc-N-DBODC5. Angiography agreement was very good for 99m Tc-N-DBODC5 ( = 0.73) and moderate for 99m Tc-MIBI ( = 0.52). Specificity and accuracy were not significantly different but better for the 99m Tc-N-DBODC5 group when compared to 99m Tc-MIBI SPECT (specificity, 88-65%; accuracy, 87-78%). Table 4 also shows the sensitivity and specificity of the two tracers in detecting individual-stenosed vessels. Of a total of 138 arteries in 46 patients, 48 arteries had significant stenoses, and 90 had insignificant lesions or were normal. Overall figures for sensitivity and specificity in identifying individualstenosed vessels were 67% (32/48) and 86% (77/90) for 99m Tc-MIBI imaging; overall sensitivity and specificity to diagnose individual significantly stenosed vessels were 69% (33/48) and 92% (83/90) for 99m Tc-N-DBODC5 SPECT studies.

Discussion
These preliminary results demonstrate that stress-rest myocardial perfusion SPECT with 99m Tc-N-DBODC5 is a sensitive method for detecting CAD and identifying stenosed coronary arteries. For the detection of CAD, more importantly, 99m Tc-N-DBODC5 MPI reached good agreement compared with CA ( = 0.73).

Advantages of 99
Tc-N-DBODC5. 99m Tc-N-DBODC5 is a new lipophilic, monocationic, and nitride 99m Tc-labeled tracer that is rapidly cleared from the liver after intravenous injection. It possesses good stability under physiological conditions. Importantly, 99m Tc-N-DBODC5 exhibits more rapid liver washout than either 99m Tc-MIBI or 99m Tc-tetrofosmin. For example, at 60 min after injection in rats, the heart/liver ratio of 99m Tc-N-DBODC5 is approximately ten times higher than that of 99m Tc-MIBI or 99m Tc-tetrofosmin. Preclinical studies have shown that 99m Tc-N-DBODC5 SPECT can identify previous ischemia as areas of reduced tracer uptake [25,26]. Furthermore, the rapid liver clearance and high uptake in the myocardium of 99m Tc-N-DBODC5 will allow Table 4: Sensitivity, specificity, and diagnostic accuracy of scintigraphic perfusion studies and agreement with coronary angiography.
Overall LAD  LCX  RCA  MIBI  DBODC  MIBI  DBODC  MIBI  DBODC  MIBI  DBODC  No. of disease  25  25  10  12  9  8  13  13  Sensitivity (%)  86  86  53  63  64  57  87  87  Specificity (%)  65  88  96  96  94  94  68  SPECT images of the left ventricle to be acquired early and with excellent quality [22]. The ratios of heart-to-liver were consistent with the previous report study [22]. These characteristics are suitable, particularly for patients with suspected acute coronary syndromes and without diagnostic electrocardiogram, because early diagnosis is needed in such patients for timely therapeutic decision making. 99m Tc-MIBI, on the other hand, may require a wait time of up to 1 h for imaging.
In the present study, the heart-to-organ count ratio of 99m Tc-N-DBODC5 MPI was superior to that of 99m Tc-MIBI in 46 patients. Compared with 99m Tc-MIBI MPI, 99m Tc-N-DBODC5 MPI had better image quality in most patients. Furthermore, in this study, exercise stress myocardial images were performed 30 min after 99m Tc-N-DBODC5 injection, and excellent MPI with high contrast was possible.

Clinical Value of 99
Tc-N-DBODC5 for the Detection of CAD. This clinical trial assesses the diagnostic value of this agent to detect CAD comparing it with stress 99m Tc-MIBI MPI and CA.
In this study, compared with 99m Tc-MIBI MPI, 99m Tc-N-DBODC5 MPI indicates the same sensitivity and better specificity and accuracy to detect coronary disease in patients, although none of the differences were significant. On the other hand, more importantly, angiography agreement was very good for 99m Tc-N-DBODC5 ( = 0.73) and moderate for 99m Tc-MIBI ( = 0.52); thus, compared with 99m Tc-MIBI MPI, it provides a high degree of concordance for the evaluation of CAD, specifically in excluding perfusion abnormalities in patients with suspected CAD. Possible reasons for this difference may be fast liver clearance, favorable heartto-organ ratio, and high image quality. 99m Tc-N-DBODC5 with rapid liver clearance may significantly reduce the photon scatter from the liver into the inferoposterior walls. This reduces the artifactual decreased myocardial perfusion and improves the diagnostic accuracy for the detection of CAD compared with other 99m Tc-labeled perfusion agents [24].
Unsurprisingly, Braat et al. [35] and Germano et al. [36] noted that technetium-labeled MPI agents with a fast liver clearance can significantly reduce the photon scatter from the liver into the inferior walls, but the radioactivity may be transferred to the gastrointestinal area, and increased bowel and gastric activity were seen as a problem associated with high liver uptake in the visual and quantitative interpretation of the inferoposterior myocardial walls. In this study, bowel uptake was frequently seen on the 99m Tc-N-DBODC5 images, but did not result in any nondiagnostic scans.
For the detection of coronary disease in patients, 99m Tc-MIBI produced abnormal results for MPI in four patients who had no angiographically detected stenosis. Some of these false-positive results may be due, in part, to the liver-toheart artifacts. In this study, we could classify fixed perfusion defects as soft-tissue attenuation artifacts or infarcts by using gated SPECT. Because an artifactual defect would show normal contraction (wall motion or thickening) on a gated image, artifacts can be differentiated from a true infarct [37].
For the detection of individual vessel stenosis, on the other hand, angiography agreement was very good for 99m Tc-N-DBODC5 ( = 0.71 for RCA) and moderate for 99m Tc-MIBI ( = 0.48 for RCA). For LAD and LCX arteries stenosis, the diagnosis of myocardial ischemia on 99m Tc-N-DBODC5 and 99m Tc-MIBI study had the same specificity; and it was similar to that of two tracer to diagnose sensitivity and accuracy of myocardial ischemia. Abnormal results of myocardial images in individual RCA vessel stenosis (six arteries-stenosed vessels for RCA) were obtained in the 99m Tc-MIBI studies, while CA and 99m Tc-N-DBODC5 studies showed normal findings in the same individual RCA vessel territories. Some of these false-positive results may be due to photon scatter from the liver and intestine into the inferoposterior walls. 99m Tc-MIBI concentration located below the left diaphragm (i.e. liver and bowel) may cause an artifactual perfusion defect in the adjacent myocardial, a phenomenon known as the "liver-heart artifact" [36,38]. Moreover, the inferior and inferoposterior regions were dominated mainly by RCA territories corresponding to the myocardial short axis and vertical long axis; while arteries regions which LAD and LCX dominated were less interfered from "liver-heart artifact. " Finally, for clinical applications, 99m Tc-N-DBODC5 offers better determination of detects, particularly in the inferoposterior wall on myocardial images. A typical example of a false-positive perfusion defect in the inferior wall is shown in Figure 5.
On a segment-to-segment basis, complete agreement between the two imaging agents occurred in 71% of segments.  Figure 5: Serial short-axis and vertical long-axis slices of stress-rest 99m Tc-N-DBODC5 images (b) and stress-rest 99m Tc-MIBI images (c) of a representative patient with normal CA (a). Because of intense uptake of technetium 99m Tc-MIBI in the liver, high liver background activity can be observed. Furthermore, a false-positive myocardial perfusion defect was also seen in the inferoposterior wall segments supplied by the RCA territory (white arrows). Importantly, however, at stress and rest, the inferoposterior wall segments of 99m Tc-N-DBODC5 images are clearly separated from the subdiaphragmatic activity.
However, more myocardial segments were nonreversible on 99m Tc-MIBI images than on 99m Tc-N-DBODC5 images. On the contrary, 99m Tc-N-DBODC5 MPI had a higher reversible defect than that of 99m Tc-MIBI. A possible reason for this difference may be due, in part, to the "liver-heart artifact. " The presence of high liver activity adjacent to the inferior wall results from oversubtraction of activity from the inferior wall. Therefore, the more "liver-heart artifact" in the inferoposterior wall on myocardial images, the more false nonreversible defects in the inferoposterior wall on myocardial perfusion segmental analysis. In other words, the less reversible defects in the inferoposterior wall were deduced on 99m Tc-MIBI MPI segmental analysis. More importantly, however, the main advantage and clinical value of 99m Tc-N-DBODC5 can improve diagnostic specificity and reduce the false-positive diagnosis of patients and associated treatment fees.

Limitations
In this study, we did not attempt to assess the absolute diagnostic accuracy of 99m Tc-N-DBODC5, because definitive conclusions in this regard can only be drawn by studying larger patient cohort. In addition, although 99m Tc-N-DBODC5 did not track flow as well as 201 Tl, the magnitudes of the pharmacologic stress-induced perfusion defects were comparable to those previously reported for 99m Tctetrofosmin. Thus, direct comparison between 99m Tc-N-DBODC5 and 99m Tc-tetrofosmin should be further proved in the same patient population. Overall, 99m Tc-N-DBODC5 MPI will become an important diagnostic tool in the evaluation of myocardial perfusion.

Conclusion
This preliminary clinical study showed that 99m Tc-N-DBODC5 and 99m Tc-MIBI MPI provide comparable diagnostic information for patients undergoing exercise rest for detection of CAD. In addition, 99m Tc-N-DBODC5 does not exhibit the disadvantages of 99m Tc-MIBI in this study. By contrast, because of its high heart-organ count ratio in comparison to 99m Tc-MIBI, it improves high degree of diagnostic concordance in defining or excluding perfusion abnormalities in patients with CAD. Therefore, it can become the agent of choice for the evaluation of myocardial perfusion and ventricular function in patients with CAD.

Ethics Statement
This study was conducted in accordance with the Helsinki Declaration. All subjects provided written informed consent before MPI, acknowledging that they understood their rights and obligations. This study was approved by the Research Ethics Committee of Shanxi Medical University.