Vitreous Fatty Amides and Acyl Carnitines Are Altered in Intermediate Age-Related Macular Degeneration

Abstract

Purpose: Age-related macular degeneration (AMD) is the leading cause of visual impairment worldwide. In this study, we aimed to investigate the vitreous humor metabolite profiles of patients with intermediate AMD using untargeted metabolomics.

Methods: We performed metabolomics using high-resolution liquid chromatography mass spectrometry on the vitreous humor of 31 patients with intermediate AMD and 30 controls who underwent vitrectomy for epiretinal membrane with or without cataract surgery. Univariate analyses after false discovery rate correction were performed to discriminate the metabolites and identify the significant metabolites of intermediate AMD. For biologic interpretation, enrichment and pathway analysis were conducted using MetaboAnalyst 5.0.

Results: Of the 858 metabolites analyzed in the vitreous humor, 258 metabolites that distinguished patients with AMD from controls were identified (P values < 0.05). Ascorbic acid and uric acid levels increased in the AMD group (all P values < 0.05). The acyl carnitines, such as acetyl L-carnitine (1.37-fold), and fatty amides, such as anandamide (0.9-fold) and docosanamide (0.67-fold), were higher in patients with intermediate AMD. In contrast, nicotinamide (−0.55-fold), and succinic acid (−1.69-fold) were lower in patients with intermediate AMD. The metabolic pathway related oxidation of branched chain fatty acids and carnitine synthesis showed enrichment.

Conclusions: Multiple metabolites related to fatty amides and acyl carnitine were found to be increased in the vitreous humor of patients with intermediate AMD, whereas succinic acid and nicotinamide were reduced, suggesting that altered metabolites related to fatty amides and acyl carnitines and energy metabolism may be implicated in the etiology of AMD.

Age-related macular degeneration (AMD) is the leading cause of visual impairment in developed countries,^1,2 particularly in elderly individuals. AMD is a severe health problem considering its high incidence, which is estimated to be approximately 8.7% worldwide.³ In addition, late-stage AMD, including neovascular AMD and atrophic AMD, is a major cause of AMD-related vision loss^4–6; hence, early detection and treatment of AMD is important. Moreover, as people with intermediate AMD are at a high risk of progressing to late AMD, as classified by the Age-Related Eye Disease Study (AREDS) grading, diagnosis in the early stage of AMD is crucial.^6,7 The greatest challenge associated with AMD lies in the absence of symptoms during the intermediate period; as patients with intermediate AMD show many medium-sized drusen or at least one large drusen in one or both eyes, as well as alterations in the retinal pigment epithelium (RPE), without meaningful visual deficits.⁸ 

The risk factors for AMD are advanced age, genetic factors, smoking, body mass index, dietary factors, and hypertension.^9–12 Dyslipidemia and metabolic dysfunction have also been found to be associated with AMD.^13,14 However, putative causal relationships have not been clearly demonstrated.¹⁵ As RPE provides metabolic support for photoreceptors and choroid cells, it is believed that the very high metabolic activity of the macula places a high cumulative demand on the RPE over the individual's lifetime for the breakdown and removal of metabolic waste products.^16,17 In this regard, the application of metabolomics in AMD would unveil candidate key metabolites related to AMD pathogenesis, because recent advances in metabolomics allow a direct signature of the metabolic state of the organism and offer important clues on the pathological conditions that cause altered concentrations of specific metabolites.^18,19 

Several studies on the metabolomics of patients with AMD have identified metabolites with altered plasma levels, such as cysteine, glycerophospholipids, carnitine, and oleic acid.^20–27 Because the eye has a blood-retinal barrier, it is preferred that intraocular fluids, such as aqueous humor and vitreous humor, are used instead of blood to better understand the metabolic alterations in the retina. It would be beneficial to compare the association between significant metabolites in the blood and intraocular fluids around the retina in patients with AMD. However, few studies have used aqueous humor or vitreous humor to investigate metabolites of AMD owing to the constraints of intraocular fluids collection. Hence, utilizing metabolome analysis on vitreous humor, we attempted to uncover metabolite alterations and key pathways in intermediate AMD in comparison to controls. To this end, we conducted high-resolution untargeted metabolomics and pathway analysis using vitreous humor samples collected during vitrectomy for epiretinal membrane (ERM) removal in patients with intermediate AMD and controls, to help gain a better understanding of AMD. 

Methods

Study Design

The research protocol was conducted in accordance with the tenets of the Declaration of Helsinki and approved by the Seoul National University Hospital (SNUH) Institutional Review Board (IRB No. 1612-093-815). All participants provided written informed consent and were recruited prospectively from March 2017 to April 2018. We recruited participants who underwent pas plana vitrectomy for ERM at the time of SNUH retinal service. In cases where patients had cataracts, combined phacovitrectomy was performed. Undiluted vitreous humor samples (at least 1 mL) were collected during the initial dry vitrectomy. The samples were covered with early cryocoats of liquid nitrogen and then stored. Prior to enrollment, all patients underwent a retinal examination by a retinal specialist, which included slit-lamp biomicroscopy, dilated fundus examination, fundus photography, and optical coherence tomography. AMD was defined using a modified version of the AREDS grading system as follows.^6,7 No abnormal findings, no drusen, and no AMD pigmentary abnormality; normal aging change, small drusen ≤63 µm, and no AMD pigmentary abnormality; early AMD, medium drusen >63 µm and ≤125 µm, and no AMD pigmentary abnormality; intermediate AMD, large drusen >125 µm, and/or any AMD pigmentary abnormality; and late AMD, neovascular AMD, and/or geographic atrophy. Among participants, patients with intermediate AMD were included in the study, and patients with no abnormal findings, according to AREDS gradings, were assigned to the control group. Patients with other retinal diseases (retinal arterial ischemia or occlusion disease, etc.) except AMD or ERM, optic nerve diseases, glaucoma, and ischemic/inflammatory brain diseases were not included in this study. Patients <55 years old, lack of phenotype data, and insufficient volume for liquid chromatography mass spectrometry (LC-MS) measurement were excluded. Information on the patients’ age, sex, intraocular pressure, laboratory data including lipid panel, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels, smoking, alcohol, diabetes mellitus (DM), hypertension (HTN), and ischemic heart diseases, as well as a history of taking the Age-Related Eye Disease Study 2 (AREDS2) formula, was collected. 

Sample Preparation and Liquid Chromatography Mass Spectrometry

The vitreous humor samples were stored at −80°C prior to measuring their concentrations. After 100 µL of vitreous humor was combined and mixed well with 375 µL of chloroform/methanol mixture (1:2), the solution was mixed with 125 µL H₂O and 125 µL CHCL₃. After centrifugation at 8000 rpm for 20 minutes, the upper layer of the aqueous phase and lower layer of the organic phase were collected and dried using a vacuum centrifuge (Speedvac, RVC 2-25 CDplus, Martin Christ, Germany). The dried sample was hydrolyzed with 100 µL of LC mobile solution A for the aqueous phase (0.1% formic acid in water) and mobile phase B for the organic phase (0.1% formic acid in methanol). After centrifugation of samples at 14,000 rpm for 15 minutes, the supernatant was carefully moved to autosampler at 4°C using an autosampler vial. Analyses were performed using a Vanquish/Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer system (Thermo Scientific, Sunnyvale, CA, USA) along a reverse-phase column (Pursuit C18 150 mm × 2.1 mm, 3 µm) and a hydrophilic interaction liquid chromatography (HILIC) column (Zorbax HILIC plus, 100 mm × 2.1 mm, 3.5 µm). A scan range of m/z 67 to 1000 was chosen as the centroid mode, with the resolution for higher-energy collisional dissociation cell spectra set to 70,000 at m/z. Compound Discoverer software version 3.1 (Thermo Fisher Scientific) was used for the extraction and identification of metabolic features using database search, which included BioCyc, Chembank, Human Metabolome Database, Kyoto encyclopedia of genes and genomes (KEGG), and mzCloud. The metabolites with fully matched annotation were examined in order to improve the reliability. 

Statistical Analysis of Individual Metabolites

For descriptive analysis, comparisons were made between cases and controls. Based on the results of the normality test for continuous variables, it was decided whether to use a parametric or non-parametric method; two-sample t-test or Mann–Whitney U test was performed. The difference in m/z features was adjusted using the Benjamini-Hochberg correction for false discovery rate (FDR) to account for multiple comparisons.²⁸ Principal component analysis (PCA) was used to determine the similarity of metabolomics between the intermediate AMD and control groups. The statistical significance for the volcano plot was determined when the −log 10 (FDR corrected P value) with P < 0.05 on the Y-axis and the log₂ fold change on the X-axis was greater than 1 or less than −1. Statistical analyses were performed using the R 3.6.3 program (R Foundation, Vienna, Austria), and the level of statistical significance was set at P < 0.05. 

Metabolic Pathway Analysis

Metabolic pathway set enrichment analysis and pathway analysis were conducted using MetaboAnalyst 5.0 (https://www.Metaboanalyst.ca/) computational platform to better understand the functional evaluation. 

Results

Characteristics of the Study Participants

Initially, vitreous samples from 34 patients with intermediate AMD and 32 controls were collected, and 5 samples (3 patients with AMDs and 2 controls) were excluded because of insufficient volumes for preprocessing, age, or missing data. Finally, vitreous samples from 31 patients with intermediate AMD and 30 controls were analyzed in this study (Fig. 1). The clinical and baseline demographic characteristics of the study groups are presented (Table 1). The age of the patients with intermediate AMD was relative older than that of the control group (69.45 ± 6.67 vs. 66.37 ± 5.42, P = 0.053) without statistical significance. The ratios of sex, DM, HTN, ischemic heart disease/strokes, ratio of smoking habit, alcohol drinking, ALT, and AST were not different between both groups (all P values > 0.05). In addition, the number of AREDS2 formula was not statistically different between AMD subjects and control patients (entire: P = 0.466, formula A: alpha-carotene, lycopene, beta carotene, vitamin A and E, beta cryptoxanthin, iron and zinc, lutein, selenium, zeaxanthin: P = 0.941, formula B: astaxanthin, saffron, zeaxanthin, selenium, lutein, vitamin D, chlorogenic acid: P = 0.399, respectively). There were no differences in total, low-density lipoprotein, and high-density lipoprotein cholesterol levels across the lipid panels (all P values > 0.05), although there were substantial disparities in triglyceride levels (P = 0.007). 

Figure 1.

 

Schematic flow of the study. AMD, age-related macular degeneration; PPV, pas plana vitrectomy; LC-MS, liquid chromatography-mass spectrometry; ERM, epiretinal membrane *Patients with other retinal diseases (retinal arterial ischemia or occlusion disease, etc.) except AMD or ERM, optic nerve diseases, glaucoma, and ischemic/inflammatory brain diseases were not included in this study.

Table 1.

 

Baseline Characteristics of Enrolled Patients

 

Table 2.

 

Candidate Metabolites Related to Intermediate AMD from the Entire Identified Significant Metabolites with Adjustment with Age and AREDS2 Formula

 

Global (Untargeted) Metabolome Profiling for Vitreous Humor

PCA showed a trend of metabolites separated between groups of participants with intermediate AMD and controls, indicating differences among them (Supplementary Fig. S1). In addition, heatmaps for the top 100 metabolites were expressed differently between the cases and controls (Supplementary Fig. S2). Of the 858 metabolites identified in the vitreous humor, the volcano plot represented the relationship between fold change and significance of metabolic features for the statistically significant 258 metabolites with P < 0.05. In addition, 97 metabolites increased and 33 metabolites decreased when criteria included the log₂ fold change on the X-axis was greater than 1 or less than −1, for patients with intermediate AMD when compared with controls after age and AREDS2 formula adjustment (Fig. 2 and Supplementary Table S1). 

Figure 2.

 

The volcano plot representing significant metabolites in the vitreous of AMD versus control. Of the 858 metabolites analyzed in the vitreous humor, the volcano plot of differential analysis identified a set of 258 metabolites with P values < 0.05, of them, 97 metabolites increased (UP, red color) and 33 metabolites decreased (DOWN, blue color) in patients with intermediate AMD when compared with controls after age and AREDS2 formula adjustment. The statistical significance for volcano plot was determined when the −log 10 (P value) with P < 0.05 on the Y-axis and the log₂ fold change on the X-axis was greater than 1 or less than −1. AMD, age-related macular degeneration; AREDS2, Age-Related Eye Disease Study 2.

Figure 3.

 

Significant metabolites after adjustment with age and AREDS2 formula. The significant metabolites derived from mzCloud Best match >90, with annotated metabolites (see Supplementary Table S2). In patients with intermediate AMD, metabolites related fatty amides and acyl carnitines were found to be expressed at a higher level than in controls. AMD, age-related macular degeneration; AREDS2, Age-Related Eye Disease Study 2.

Significant Vitreous Humor Metabolites for Patients With Intermediate AMD

Metabolites believed to be related to the AREDS2 formula were altered in individuals with intermediate AMD compared to controls (Fig. 3, Table 2 [mzCloud > 90] and Supplementary Table S2): ascorbic acid levels (14.09-fold), uric acid (8.94-fold), L-threonic acid (4.14-fold), and coenzyme Q2 (0.91-fold). Fatty amides were significantly increased in the vitreous humor of patients with intermediate AMD: stearamide (2.02-fold), anandamide (0.90-fold), lauramide (0.81-fold), oleoylethanolamide (0.80-fold), linoleamide (0.72-fold), stearoylethanolamide (0.71-fold), docosanamide (0.67-fold), palmitoleic acid (0.60-fold), and hexadecanamide (0.53-fold). In addition, acyl carnitine was significantly highly expressed in patients with AMD: 2-methylbutyrylcarnitine (2.31-fold), acetyl L-carnitine (1.37-fold), and L-palmitoylcarnitine (1.36-fold). In contrast, citrate cycle-related metabolites, such as succinic acid (−1.69-fold), nicotinate and nicotinamide metabolism, such as nicotinamide (−0.55-fold), and endocannabinoid-like metabolites, such as N-arachidonoyl-L-serine (−0.49-fold), were decreased in patients with intermediate AMD when compared with controls. 

Enrichment Analysis and Metabolic Pathway Analysis

The set enrichment analysis showed that oxidation of branched chain fatty acid, carnitine synthesis, and mitochondria electron transport chain are related with the metabolites identified in this study (Fig. 4). In addition, caffeine metabolism and oxidation of branched chain fatty acids were significant pathways (Supplementary Table S3). 

Figure 4.

 

Enrichment analysis using Metaboanalyst 5.0 from identified metabolites. Reference database was chosen of the Small Molecule Pathway Database (SMPDB)⁵² Oxidation of branched chain fatty acid, carnitine synthesis related metabolisms were enriched.

Discussion

Our study on the vitreous humor of elderly patients with intermediate AMD demonstrated that fatty amides, such as docosanamide, and acyl carnitines, such as acetyl L-carnitine and L-palmitoylcarnitine, were significantly increased, whereas energy metabolism-related metabolites, such as succinic acid and nicotinamide, decreased. In addition, metabolites related to the AREDS2 formula and nutritional supplements, such as ascorbic acid, uric acid, and L-threonic acid, were highly increased in patients with intermediate AMD. These metabolite alterations found in patients with intermediate AMD are connected with altered lipid and energy metabolism, suggesting that this pathway modification may be associated with the pathogenesis of AMD. 

Several studies have been conducted on the plasma of patients with neovascular AMD.^24,26,29,30 It is beneficial for this study, because it can be directly related to the body's metabolism, as it can secure a large number of samples by making the blood sample considerably less invasive. However, there is a blood-retina barrier; therefore, it is difficult to directly determine the metabolism of the blood and RPE. In this respect, our work is believed to have future research significance because it uses biological fluids, such as vitreous humor, which is a valuable sample. Ascorbic acid, also known as vitamin C, is a water-soluble molecule present in most tissues in its anionic state but cannot be synthesized by humans and can be obtained exogenously. The vitreous receives its vitamin C supply from the plasma through active transport from the ciliary body, up to 33 times more than the plasma concentrations.³¹ There was a 14.09-fold rise in our study, which is likely to be connected to the usage of the AREDS2 formula. The increase in uric acid is presumed to be a result of alterations in purine metabolism associated with neurodegeneration, such as hypoxanthine in AMD development.³² Because the enrolled patients with AMD used the AREDS2 formula, which contains lutein/zeaxanthin, vitamin E, and zinc, it is challenging to rule out this impact. In addition, several controls (patients with ERM with no AMD) had taken the AREDS2 formula, according to the recommendations of retinal physicians or media promotions, with patients purchasing it from the pharmacies or home shopping and consuming it at their own will (regarding as a nutrient supplement). Considering that the AREDS2 formula is a preventative supplement and not a treatment for AMD, as they are uncontrollable aspects in clinical scenarios, these components should be considered when interpreting the significance of the data. Due to the fact that it was a relative quantitative investigation (untargeted LC-MS), both control and case groups received statistically equivalent doses, an excess was not identified. However, because it is a factor that can affect it, it was considered as an adjusting factor for statistical analysis. 

Inflammation, complement dysregulation, oxidative stress, extracellular matrix remodeling, dysregulated lipid metabolism, and angiogenesis have all been identified as key pathobiological factors causing AMD development and progression.^33,34 Although, our study did not include glaucoma and inflammatory/ischemic brain disease, these conditions would be an interesting topic of investigation for the next study. Furthermore, metabolomics and genetics research approaches that have emerged as analytical techniques have demonstrated that altered lipid metabolic pathways, including hepatic lipase (LIPC), cholesteryl ester transfer protein (CETP), and ATP-binding cassette subfamily A member 1 (ABCA1), are related to the etiology of AMD.^{9,23,24,29,35–37} In addition, our findings imply that the increase in fatty amides, including stearamide, stearoylethanolamide, anandamide, oleoylethanolamide, linoleamide, and hexadecanamide, in the vitreous humor may be implicated in the pathogenesis of AMD. Lipid metabolism has been reported as one of the major pathways affected in AMD; fatty acid, such as linoleic acid and oleic acid were increased in patients with AMD.^34,38 In addition, endocannabinoid-like metabolites and anandamide were highly elevated in the retina of post-mortem eyes, which suggests that compounds are involved in AMD pathology.³⁹ These endogenous endocannabinoid metabolites and their binding receptors have been discovered in retinal tissue and have been linked to neuroprotection and neurodegeneration.^40,41 These metabolites require additional investigation because they are linked to glaucoma, ischemia, or inflammatory brain damage, as well as AMD.⁴¹ Epidemiological data have found that high alpha-linolenic acid (trans) consumption is related to a higher incidence of intermediate AMD.⁴² This implies that an increase in either endogenous or exogenous fatty amides is linked to retinal degeneration. 

A previous study on metabolomics using plasma by Mitchell et al. showed that multiple long-chain acyl carnitines, which are part of the carnitine shuttle pathway, were significantly increased in the plasma of patients with neovascular AMD.⁴³ As a result of the esterification of L-carnitine to create acyl carnitine derivatives. The carnitine shuttle route is responsible for delivering long-chain fatty acids into the mitochondria for subsequent degradation by β-oxidation (Supplementary Fig. S3).⁴⁴ These findings were supported by another study using the aqueous humor in neovascular AMD, which showed a compromised carnitine-associated mitochondrial oxidation pathway (carnitine, deoxycarnitine, and N6-trimethyl-l-lysine).⁴⁵ According to a recent study on plasma metabolomics of intermediate AMD and neovascular AMD,²² the carnitine shuttle route was considerably different in both intermediate AMD and neovascular AMD when compared to controls, revealing that intermediate AMD and controls had limited discriminant values for the carnitine shuttle using plasma metabolomics. In this regard, the findings of our study are noteworthy because the carnitine shuttle pathway, such as L-palmitocarnitine, was identified by examining the vitreous humor for intermediate AMD (see Supplementary Fig. S3 in KEGG for pathway of metabolites), since observing the same trend in ocular fluid and plasma provides crucial information (Table 3). 

Table 3.

 

Comparison of Identified Metabolites in Plasma, Aqueous Humor, and Vitreous Humor in Intermediate Age-Related Macular Degeneration

 

In our study, the levels of energy metabolism-related metabolites, such as succinic acid and nicotinamide, decreased. These results are consistent with those of a previous meta-analysis of AMD cohorts,²⁶ which showed that tricarboxylic acid (TCA) cycle metabolites, glycerophospholipid metabolites, and nicotinate and nicotinamide metabolisms were significantly different. Succinate is a key intermediate of the TCA cycle that donates electrons to the electron transfer chain, which is essential for the production of adenosine triphosphate in mitochondria.⁴⁶ Nicotinamide is a precursor of nicotinamide adenine dinucleotide (NAD), which has been postulated to ameliorate AMD metabolism in experimental studies using human-induced pluripotent stem cell (RPE)-based AMD models,^47,48 providing evidence that nicotinamide may be a potential therapeutic target for AMD. 

An advantage of this study was that it was not evaluated using blood but rather vitreous samples that revealed innate metabolites. Because vitreous humor sampling is invasive, studies on retinal detachment and diabetic retinopathy have been conducted^49,50; however, there are a few studies on the metabolomics of intermediate AMD. In addition, our study focused on early and intermediate AMD, which gives it an edge in the early identification of metabolites compared to other studies that have addressed neovascular AMD. Nevertheless, this study had several limitations. First, a cross-sectional study uses an untargeted metabolomics approach for screening; as targeted metabolomics may offer quantitative information, it is better to combine the two methods concurrently if the sample amount is unlimited. Further studies are necessary for targeted validation, and targeted measured analysis would be helpful outside this cohort. Given this point in the analysis of the results, metabolites selection is believed to be required. Second, considering that genetic variations can affect both lipid levels and AMD risk, we did not include this effect because we did not analyze patient genetic background, which limits the interpretation of the results. In future research, it is anticipated that results with high internal validity will be obtained if multi-omics analysis results are provided. In addition, increased or reduced metabolites may be the cause of AMD pathology; however, they may also be the result of RPE destruction due to AMD, necessitating the need for more experimental research. Third, targeting 31 individuals with intermediate AMD is not a small number, although it is predicted that a larger target group would yield better outcomes. In addition, some researchers anticipate discovering more significant metabolomes by comparing late and intermediate AMD. However, research design should be evaluated in light of circumstances beyond the analysis laboratory. In clinical situations when vitreous was collected by pas plana vitrectomy (PPV) in order to remove ERM, retinal surgery was conducted when it was expected to improve the patient's visual acuity. As late AMD comprises atrophic AMD and neovascular AMD: (1) even if ERM is surgically removed, atrophic AMD does not have a good prognosis for recovery of visual acuity, and (2) neovascular AMD will produce multiple metabolites as a result of blood itself; our study was developed for early type AMD to minimize confounding factors. Fourth, it would be an excellent research if fasting plasma data were collected with vitreous samples; however, unfortunately, this was not the case. It is believed that a comparative verification research using blood-based metabolites and eye-derived fluids collected at the same time will aid in the development of a potential biomarker if conducted as part of the follow-up investigation. According to a previous study,⁴³ carnitine shuttle in blood was replicated in our study in vitreous humor samples (see Table 3), indicating that the metabolite had a potential role in the pathogenesis of AMD. 

In conclusion, altered metabolism-related fatty amides and acyl carnitines were found in the vitreous humor of the elderly with intermediate AMD. In addition, energy metabolism-related metabolites, such as nicotinamide and succinic acid, decreased. These altered lipid metabolism and energy metabolism might be associated with the pathogenesis of intermediate AMD. Further studies are required to determine the causality between these metabolites. 

Acknowledgments

Supported by the Veterans Health Service Medical Center Research Grant (grant number: VHSMC190025) and National Research Foundation of Korea (NRF) grant funded by the Korean government (Ministry of Science and ICT; No. 2022R1C1C1002929), in part, by grants from the Korean Association of Retinal Degeneration (grant number: KARD2021001) and “Korea Research-Driven Hospital” (grant number: HI14C1277) through the Korea Health Industry Development Institute, funded by the Ministry of Health and Welfare, Korea. 

MetaboAnalysit 5.0: (https://www.metaboanalyst.ca/MetaboAnalyst/). 

KEGG Database Project: (Kyoto Encyclopedia of Genes and Genomes): Kanehisa Laboratories for Fatty acid Metabolism and Fatty acid Degradation and Nicotinate and Nicotinamide Metabolism (map00071, map01212, hsa00061, hsa00760, permission:230103 and 230394).⁵¹ 

LIPIDMAPS: (https://www.lipidmaps.org/). 

Human Metabolome Database: (HMDB; https://hmdb.ca/). 

Small Molecule Pathway Database: (SMPDB; https://www.smpdb.ca/).⁵² 

Ethical Approval: This study protocol was approved by the Institutional Review Board of Seoul National University Hospital (IRB No. 1612-093-815). 

Informed Consent: A written informed consent form was obtained by each patient before starting the study. 

Availability of Data and Material: The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request. 

Disclosure: C.-K. Yoon, None; Y.A. Kim, None; U.C. Park, None; S. Kwon, None; Y. Lee, None; H.J. Yoo, None; J.H. Seo, None; H.G. Yu, None