Prediction of sarcopenia using a combination of multiple serum biomarkers

Prediction of sarcopenia using a combination of multiple serum biomarkers www.nature.com/scientificreports OPEN Prediction of sarcopenia using a combination of multiple serum biomarkers Received: 22 November 2017 1 2 3 1 1 Ju Y eon Kwak , Hyeoncheol Hwang , Seon-Kyu Kim , Jeong Yi Choi , Seung-Min Lee , 2 1 1,5 2,4 1,6 Accepted: 15 May 2018 Hyun Bang , Eun-Soo Kwon , Kwang-Pyo Lee , Sun Gun Chung & Ki-Sun Kwon Published: xx xx xxxx Sarcopenia is a gradual loss of skeletal muscle mass and function with aging. Given that sarcopenia has been recognized as a disease entity, effective molecular biomarkers for early diagnosis are required. We recruited 46 normal subjects and 50 patients with moderate sarcopenia aged 60 years and older. Sarcopenia was clinically identified on the basis of the appendicular skeletal muscle index by applying cutoff values derived from the Asian Working Group for Sarcopenia. The serum levels of 21 potential biomarkers were analyzed and statistically examined. Interleukin 6, secreted protein acidic and rich in cysteine, macrophage migration inhibitory factor, and insulin-like growth factor 1 levels differed significantly between the normal and sarcopenia groups. However, in each case, the area under the receiver operating characteristics curve (AUC) was <0.7. Subsequent combination of the measurements of these biomarkers into a single risk score based on logistic regression coefficients enhanced the accuracy of diagnosis, yielding an AUC value of 0.763. The best cutoff value of 1.529 had 70.0% sensitivity and 78.3% specificity (95% CI = 2.80–21.69, p < 0.0001). Combined use of the selected biomarkers provides higher diagnostic accuracy than individual biomarkers, and may be effectively utilized for early diagnosis and prognosis of sarcopenia. Sarcopenia, defined as age-related decline in skeletal muscle mass and function , is the most significant cause of frailty in the elderly . e Th recent upsurge of research on sarcopenia has supported the establishment of a new dis- ease code in ICD-10-CM . Therefore, effective diagnostic tools that could facilitate early diagnosis and prognosis of sarcopenia remain an urgent unmet medical need. Diagnostic criteria for sarcopenia are based on low muscle mass and function (strength or performance) . A number of technologies are currently employed to estimate muscle mass, including magnetic resonance imaging (MRI), computed tomography (CT), dual energy X-ray absorptiometry scans (DXA) and bioelectrical impedance analysis (BIA). Imaging-related quantification using MRI and CT has been considered as gold standard to measure muscle mass, but have drawbacks of high cost, low accessibility and CT-generated radiation exposure . DXA is widely used due to minimal radiation exposure and lower cost than MRI and CT . BIA is simple and portable, in spite of less reliability than imaging modalities owing to the inue fl nce of various factors, such as ethnicity, gender, age, and hydration status . Physical function tests, such as grip strength and gait speed, may be disrupted by comorbidities, such as joint problems and neuro- logic deficits, which prevail in population with sarcopenia. In addition, current methods to diagnose sarcopenia are operational only after the onset or progression of disease (e.g., loss of muscle mass or mobility disability). Therefore, determination of efficient molecular biomarkers for early diagnosis and prognosis of sarcopenia in routine clinical practice is essential . Aging Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea. Department of Rehabilitation Medicine, Seoul National University College of Medicine, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul, 03080, Republic of Korea. Personalized Genomic Medicine Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea. Institute of Aging, Seoul National University, 101 Daehak-ro, Jongno-gu, Seoul, 03080, Republic of Korea. Department of Biomolecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, 34113, Republic of Korea. Department of Functional Genomics, KRIBB School of Bioscience, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, 34113, Republic of Korea. Ju Yeon Kwak, Hyeoncheol Hwang and Seon-Kyu Kim contributed equally to this work. Correspondence and requests for materials should be addressed to S.G.C. (email: suncg@snu.ac.kr) or K.-S.K. (email: kwonks@kribb.re.kr) Scientific REPO R tS | (2018) 8:8574 | DOI:10.1038/s41598-018-26617-9 1 www.nature.com/scientificreports/ Several mechanisms are potentially involved in pathogenesis of sarcopenia. Both intrinsic (e.g., inflammation, apoptosis, autophagy, mitochondria, neuromuscular junction, calcium metabolism) and extrinsic (e.g., endo- 9–14 crine, nutritional status, immobility) factors contribute to defective myogenesis, muscle atrophy and weakness. Considering the multi-layered mechanisms underlying sarcopenia, no single biomarker may accurately represent this state. Here, we performed a comparative analysis of potential biomarkers between sarcopenia and normal elderly groups with the aim of identifying multiple serum biomarkers. We further combined the measurements of multiple biomarker candidates via logistic regression to improve diagnostic accuracy for sarcopenia. Materials and Methods Subjects and samples. Eligible candidates were community-dwelling adults aged 60 years and older. Candidates were recruited from several senior centers or halls for the elderly in Seoul. Individuals with a history of alcohol abuse or drug overuse, insulin-dependent diabetes mellitus, phenylketonuria, current heavy smokers (>1 pack/day) and amputees were excluded from study. Appendicular skeletal muscle mass (ASM) was measured using bioelectrical impedance analysis (BIA). Grip strength of the dominant hand and 4 m walking speed were additionally evaluated. Subjects were clinically classified as moderate sarcopenia according to appendicular skeletal muscle index 2 2 (ASMI; ASM/height2) <7.0 kg/m for men and <5.7 kg/m for women, as recommended by the Asian Working Group for Sarcopenia (AWGS) . In total, 50 sarcopenic and 46 normal subjects participated in the study. Blood samples were collected in serum separator tubes, frozen and stored until biomarker analysis. e I Th nstitutional Review Board of Seoul National University Hospital approved all procedures, and all meth- ods were performed in accordance with the relevant guidelines and regulations. All participants provided written informed consent upon enrollment. Enzyme-linked immunosorbent assay (ELISA). Serum levels of human IL-6, SPARC, MIF, and IGF-1 were measured using ELISA kits according to the manufacturer’s protocol (R&D systems, Inc., Minneapolis, MN, USA). Development of risk score. To develop a single risk score using all four biomarkers (IL-6, SPARC, MIF and 17,18 IGF-1), we adopted a previously developed strategy with regression analysis for multiple biomarkers . Briefly, serum levels were log transformed to reduce variations between the concentrations of each biomarker and used to generate logistic regression coefficients. The risk score for each individual was calculated as the sum of the risk score for each biomarker, which was derived by multiplying the serum level of a biomarker by its correspond- ing coefficient (Risk score = ∑ logistic regression coefficient of biomarker M × serum level of biomarker M ). i i Subjects were subsequently divided into two groups (high and low risk of sarcopenia) using the median cuto ff risk score as a threshold. Statistics. The significance of association between a risk group and clinically manifested sarcopenia was assessed using Fisher’s exact test. To estimate the utility of risk score as a diagnostic tool of sarcopenia, we cal- culated the area under curve (AUC) value using receiver operating characteristics (ROC) analysis. The signifi - cance of serum level differences between normal and sarcopenia groups was assessed using two-sample t-tests for each biomarker. Data were considered statistically significant at p-values < 0.05 and false discovery rate <0.3. All statistical analyses were performed using GraphPad Prism5 (GraphPad Software, Inc., USA) and R language environment (ver. 3.2.5). Results Baseline characteristics. Mean appendicular skeletal muscle index (ASMI) values of normal (46 sub- 2 2 2 jects) and sarcopenia (50 subjects) groups were 7.50 kg/m and 6.51 kg/m respectively in men, and 6.48 kg/m and 5.23 kg/m respectively in women. The sarcopenia group had weaker grip strength than the normal group (27.83 kgF vs 32.28 kgF in men and 17.06 kgF vs 20.86 kgF in women) but displayed no significant differences in gait speed. The baseline characteristics are shown in Table  1. Selection of biomarkers for diagnosis of sarcopenia. To identify effective molecular biomarkers for diagnosis of sarcopenia, candidates were selected by searching the relevant literature and subjected to immu- nochemical analyses using elderly people sera of normal and sarcopenia group that was clinically manifested based on AWGS. We examined 21 biomarker candidates, including proteins related to skeletal muscle function or metabolism and muscle-derived cytokines (myokines), angiotensin-converting enzyme (ACE) , insulin-like 20 21 growth factor 1 (IGF-1) , procollagen type III N-terminal peptide (P3NP) , fibroblast growth factor 21 22 23 24 25 (FGF21) , myostatin , growth differentiation factor 11 (GDF11) , meteorin-like (METRNL) , macrophage 26 27 28 migration inhibitory factor (MIF) , fatty acid binding protein 3 (FABP3) , ciliary neurotrophic factor (CNTF) , secreted protein acidic and rich in cysteine (SPARC) , brain-derived neurotrophic factor (BDNF), tumor necro- sis factor-α (TNF-α), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 15 (IL-15), interleukin 1β (IL-1β), monocyte chemotactic Protein 1 (MCP-1), transforming growth factor β 1 (TGFβ1), and vascular endothelial growth factor (VEGF) . Among the candidates investigated, serum levels of IL-6, SPARC, MIF and IGF-1 were significantly different between normal and sarcopenia groups (Fig. 1), but not the other biomarkers (data not shown). Serum lev- els of IL-6 were higher in the sarcopenia than the normal group (Fig. 1a; 1.64 ± 0.36 versus 0.86 ± 0.25 pg/ml, p = 0.015). Exceptionally, the IL-6 levels in three sarcopenia and six normal samples were undetectable with a commercially available ELISA kit that can detect concentrations as low as 0.1 pg/mL. Similarly, serum concen- trations of SPARC and MIF were higher in the sarcopenia than the normal group (Fig. 1b,c; 531.5 ± 40.65 versus 409.4 ± 35.47 pg/mL SPARC, p = 0.047, and 25.10 ± 1.19 versus 20.71 ± 0.89 ng/mL MIF, p = 0.008). Conversely, Scientific REPO R tS | (2018) 8:8574 | DOI:10.1038/s41598-018-26617-9 2 www.nature.com/scientificreports/ Men Women * * Normal Sarcopenia P-value Normal Sarcopenia P-value Number of subjects 18 18 28 32 ** Age (years) 76.00 ± 5.72 76.22 ± 5.62 0.907 71.86 ± 6.01 76.13 ± 6.05 0.008 ** Height (cm) 164.16 ± 5.02 161.56 ± 3.97 0.093 152.90 ± 5.27 147.14 ± 5.14 <0.001 ** Weight (kg) 65.68 ± 6.05 57.47 ± 6.29 <0.001 62.40 ± 6.58 49.85 ± 5.95 <0.001 ** Appendicular skeletal muscle mass (kg) 20.23 ± 2.09 16.97 ± 1.86 <0.001 15.23 ± 1.67 11.38 ± 1.29 <0.001 2 ** Appendicular skeletal muscle index (km/m ) 7.50 ± 0.41 6.51 ± 0.54 <0.001 6.48 ± 0.39 5.23 ± 0.30 <0.001 ** Grip strength (kgF) 32.28 ± 6.50 27.83 ± 5.09 0.029 20.86 ± 4.91 17.06 ± 2.71 <0.001 ** Gait speed (m/s) 0.99 ± 0.14 1.01 ± 0.16 0.685 1.00 ± 0.22 0.98 ± 0.15 0.741 Table 1. Baseline characteristics of the samples. *Independent t-test. **Mean ± standard deviation. Figure 1. Comparison of serum protein levels between normal and sarcopenic aged subjects. IL-6 (a), SPARC (b), MIF (c) and IGF-1 (d) protein levels in human serum were measured using sandwich ELISA. Box plots were used to visualize distribution of each serum protein level. P-values were obtained with two sample t-tests. serum IGF-1 levels were lower in the sarcopenia group (Fig. 1d; 58.16 ± 3.37 versus 72.61 ± 5.49, p = 0.039). Based on the collective results, IL-6, SPARC, MIF and IGF-1 were selected for analysis as potential biomarkers for sarcopenia. Application of risk score to predict sarcopenia. Combination of measurements of multiple biomarkers into a single score was required to integrate the collected information for easy diagnostic judgement. A risk score was generated by combination of the measurements of the four selected biomarkers (IL-6, SPARC, MIF and IGF-1) based on the logistic regression coefficient of each biomarker (Table  2). Using the median cutoff value of risk scores for all subjects (1.518), two groups with high-risk and low-risk scores were classified (Fig.  2a). The frequency of clinically identified sarcopenia was significantly higher in the high-risk than the low-risk group Scientific REPO R tS | (2018) 8:8574 | DOI:10.1038/s41598-018-26617-9 3 www.nature.com/scientificreports/ Protein Estimated β Std. Error P-value IL-6 0.151 0.062 0.016 SPARC 0.102 0.05 0.047 MIF 0.279 0.102 0.008 IGF-1 −0.129 0.062 0.041 Table 2. Univariate logistic regression analyses of 4 proteins. β indicates regression coefficient value. (Fig. 2b; p = 0.002, 95% confidence interval (CI) = 1.56–11.59), indicating that biomarker analysis is clinically valuable for sarcopenia diagnosis. When adapted to groups of men and women separately, biomarker analysis was successfully applied to identify sarcopenia in both genders, representing no gender differences in positive predictive value (p = 0.001, 95% CI = 2.28–171.69 in men; p = 0.009, 95% CI = 1.28–15.66 in women; Fig. 2c,d). However, the frequency of sarcopenia in the low-risk group was lower in men than women, signifying that the negative predictive value is higher in men. Significance of multiple biomarkers for diagnosis of sarcopenia. To establish whether the risk score based on multiple biomarkers is useful for diagnosis of sarcopenia, area under the ROC curve, sensitivity and specificity were assessed. When applying ROC analysis to risk score, significantly high AUC values were obtained for all population categories (AUC > 0.7). AUC was 0.763 in the overall elderly population and the best cuto ff value, defined as the point with highest multiplicity of sensitivity and specificity, was a risk score of 1.529 (Fig.  3a; 70% sensitivity and 78.26% specificity). AUC was 0.812 and the best cutoff value in elderly men (Fig.  3b; 88.88% sensitivity, 77.77% specificity) was a risk score of 1.543. In elderly women, AUC was 0.739 and a risk score of 1.505 (Fig. 3c; 68.75% sensitivity, 75% specificity) was the best cutoff value. To ascertain whether the optimal cutoff value based on ROC analysis improves predictability, subjects were dichotomized into two groups based on the above cutoff values of risk score. When applying the cutoff values of 1.529, 1.543, and 1.505 to the whole population, men and women groups, respectively, performance in classifying the high-risk sarcopenia group was improved (Supplementary Fig. 1). Our results collectively suggest that the risk score based on multiple biomark- ers selected in this study demonstrates predictive value for sarcopenia regardless of gender. Discussion While several candidate molecular biomarkers of sarcopenia have been proposed to date, none have been assessed for predictive ability. The four biomarkers analyzed in this study were not appropriate as single biomarkers for diagnosis of sarcopenia. Despite differences in serum levels between normal and sarcopenia groups with accept- able p-values (Fig. 1), none attained optimal cutoff values at which both sensitivity and specificity were > 70%, resulting in AUC <0.7 (data not shown). Interestingly, however, combination of the biomarker measurements into a single risk score enhanced the accuracy of diagnosis for sarcopenia. Sarcopenia group in our study involved elderly people with lower muscle mass (on the basis of AWGS), but excluded subjects with severe muscle weakness (grip strength <26 kgF for men and <18 kgF for women and gait speed <0.8 m/s) and potential accompanying diseases. This ‘moderate sarcopenia’ group showed slightly weaker muscle strength than the normal group but greater strength than the standard sarcopenia group (Table 1). er Th efore, selected multiple biomarkers in combination may be effectively utilized for early diagnosis of sarcope- nia before clinical manifestation of symptoms, such as weakness, disability, falls, loss of independence, and frailty. The above biomarkers are evidently correlated with skeletal muscle metabolism and function. IL-6, a well-known pro-inflammatory cytokine, was the first identified myokine . Earlier studies have reported that serum IL-6 is elevated chronically in cachexia and Duchenne muscular dystrophy (DMD) patients and acutely aer ex ft ercise, and decreases rapidly within an hour to pre-exercise levels . Serum IL-6 has been highlighted as a potential sarcopenia biomarker by several groups, but is yet to be statistically validated. SPARC, also known as osteonectin or basement-membrane protein 40, is an acidic extracellular matrix gly- 32–34 coprotein secreted by most tissues and involved in bone mineralization and cell-matrix interactions . SPARC is highly expressed in patients with various types of myopathy including DMD, inclusion body myositis, and congenital muscular dystrophy . Controversially, another study showed that SPARC overexpression extracellu- 36 37 larly inhibits muscle cell die ff rentiation , and its deficiency in mouse skeletal muscle causes myob fi er atrophy . Although reduced levels of SPARC in aged muscle may correlate with sarcopenia , changes in the circulating levels of SPARC with aging have not been documented. MIF, a pro-inflammatory cytokine, has been implicated in muscle damage, in view of its increased levels in 26 38 muscle of polymyositis patients . Since MIF is suggested to regulate glucose homeostasis and skeletal muscle is a major organ for glucose utilization, we speculate that circulating MIF levels may reflect the status of glucose metabolism in sarcopenia. IGF-1, also known as somatomedin C, is a well-known mediator of muscle growth and subsequent regenera- tion . IGF-1 signaling involving the activation of phosphatidylinositol-3-kinase and Akt (or protein kinase B) not only induces muscle hypertrophy via stimulation of protein synthesis pathways but also predominantly prevents activation of muscle atrophy pathways via inhibition of forkhead box O (FoxO) transcription factors . IGF-1 is produced by satellite cells in regenerating skeletal muscles and muscle-specific expression of the protein accel- erates regeneration of injured muscle by rapidly modulating the inflammatory response . Furthermore, IGF-1 triggers human myotube hypertrophy by accelerating the recruitment of reserve cells in human skeletal muscle . Scientific REPO R tS | (2018) 8:8574 | DOI:10.1038/s41598-018-26617-9 4 www.nature.com/scientificreports/ Figure 2. Risk score based on combination of four biomarkers (IL6, SPARC, MIF, and IGF-1) and frequency of sarcopenia. Bar graph of risk scores in 96 samples (a). Median risk score across samples was applied as a threshold for dividing into two risk groups (cutoff value = 1.518). Comparison of frequency of sarcopenia between low-risk and high-risk groups in whole population (b), Men (c), Women (d). P-values and confidence intervals (CI) were calculated with Fisher’s exact test. Figure 3. Significance of risk score based on multiple biomarkers for diagnosis of sarcopenia. Receiver operating characteristic (ROC) curves for each group. Whole population (a), Men (b), Women (c). The area under the ROC curve (AUC) was calculated for each group to determine the significance of multiple biomarkers in predicting sarcopenia. Effective multiple biomarkers for sarcopenia diagnosis are not confined to the four biomarkers assessed in this study. To reflect entire biological pathways associated with sarcopenia disease activity, optimal combinations of additional novel biomarkers require exploration. Furthermore, longitudinal studies are essential to determine whether analysis of multiple biomarkers can be employed for not only early diagnosis but also evaluating ther- apeutic interventions and prognosis prior to changes in muscle mass, strength or function. 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J.Y.K. and H.H. performed experiments and S.-K.K. calculated risk score and conducted statistical analyses. J.Y.K., H.H., S.-K.K., S.G.C., and K.-S.K. wrote the manuscript and all authors analyzed and discussed the results and edited the manuscript. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-26617-9. Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. 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Prediction of sarcopenia using a combination of multiple serum biomarkers

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www.nature.com/scientificreports OPEN Prediction of sarcopenia using a combination of multiple serum biomarkers Received: 22 November 2017 1 2 3 1 1 Ju Y eon Kwak , Hyeoncheol Hwang , Seon-Kyu Kim , Jeong Yi Choi , Seung-Min Lee , 2 1 1,5 2,4 1,6 Accepted: 15 May 2018 Hyun Bang , Eun-Soo Kwon , Kwang-Pyo Lee , Sun Gun Chung & Ki-Sun Kwon Published: xx xx xxxx Sarcopenia is a gradual loss of skeletal muscle mass and function with aging. Given that sarcopenia has been recognized as a disease entity, effective molecular biomarkers for early diagnosis are required. We recruited 46 normal subjects and 50 patients with moderate sarcopenia aged 60 years and older. Sarcopenia was clinically identified on the basis of the appendicular skeletal muscle index by applying cutoff values derived from the Asian Working Group for Sarcopenia. The serum levels of 21 potential biomarkers were analyzed and statistically examined. Interleukin 6, secreted protein acidic and rich in cysteine, macrophage migration inhibitory factor, and insulin-like growth factor 1 levels differed significantly between the normal and sarcopenia groups. However, in each case, the area under the receiver operating characteristics curve (AUC) was <0.7. Subsequent combination of the measurements of these biomarkers into a single risk score based on logistic regression coefficients enhanced the accuracy of diagnosis, yielding an AUC value of 0.763. The best cutoff value of 1.529 had 70.0% sensitivity and 78.3% specificity (95% CI = 2.80–21.69, p < 0.0001). Combined use of the selected biomarkers provides higher diagnostic accuracy than individual biomarkers, and may be effectively utilized for early diagnosis and prognosis of sarcopenia. Sarcopenia, defined as age-related decline in skeletal muscle mass and function , is the most significant cause of frailty in the elderly . e Th recent upsurge of research on sarcopenia has supported the establishment of a new dis- ease code in ICD-10-CM . Therefore, effective diagnostic tools that could facilitate early diagnosis and prognosis of sarcopenia remain an urgent unmet medical need. Diagnostic criteria for sarcopenia are based on low muscle mass and function (strength or performance) . A number of technologies are currently employed to estimate muscle mass, including magnetic resonance imaging (MRI), computed tomography (CT), dual energy X-ray absorptiometry scans (DXA) and bioelectrical impedance analysis (BIA). Imaging-related quantification using MRI and CT has been considered as gold standard to measure muscle mass, but have drawbacks of high cost, low accessibility and CT-generated radiation exposure . DXA is widely used due to minimal radiation exposure and lower cost than MRI and CT . BIA is simple and portable, in spite of less reliability than imaging modalities owing to the inue fl nce of various factors, such as ethnicity, gender, age, and hydration status . Physical function tests, such as grip strength and gait speed, may be disrupted by comorbidities, such as joint problems and neuro- logic deficits, which prevail in population with sarcopenia. In addition, current methods to diagnose sarcopenia are operational only after the onset or progression of disease (e.g., loss of muscle mass or mobility disability). Therefore, determination of efficient molecular biomarkers for early diagnosis and prognosis of sarcopenia in routine clinical practice is essential . Aging Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea. Department of Rehabilitation Medicine, Seoul National University College of Medicine, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul, 03080, Republic of Korea. Personalized Genomic Medicine Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea. Institute of Aging, Seoul National University, 101 Daehak-ro, Jongno-gu, Seoul, 03080, Republic of Korea. Department of Biomolecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, 34113, Republic of Korea. Department of Functional Genomics, KRIBB School of Bioscience, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, 34113, Republic of Korea. Ju Yeon Kwak, Hyeoncheol Hwang and Seon-Kyu Kim contributed equally to this work. Correspondence and requests for materials should be addressed to S.G.C. (email: suncg@snu.ac.kr) or K.-S.K. (email: kwonks@kribb.re.kr) Scientific REPO R tS | (2018) 8:8574 | DOI:10.1038/s41598-018-26617-9 1 www.nature.com/scientificreports/ Several mechanisms are potentially involved in pathogenesis of sarcopenia. Both intrinsic (e.g., inflammation, apoptosis, autophagy, mitochondria, neuromuscular junction, calcium metabolism) and extrinsic (e.g., endo- 9–14 crine, nutritional status, immobility) factors contribute to defective myogenesis, muscle atrophy and weakness. Considering the multi-layered mechanisms underlying sarcopenia, no single biomarker may accurately represent this state. Here, we performed a comparative analysis of potential biomarkers between sarcopenia and normal elderly groups with the aim of identifying multiple serum biomarkers. We further combined the measurements of multiple biomarker candidates via logistic regression to improve diagnostic accuracy for sarcopenia. Materials and Methods Subjects and samples. Eligible candidates were community-dwelling adults aged 60 years and older. Candidates were recruited from several senior centers or halls for the elderly in Seoul. Individuals with a history of alcohol abuse or drug overuse, insulin-dependent diabetes mellitus, phenylketonuria, current heavy smokers (>1 pack/day) and amputees were excluded from study. Appendicular skeletal muscle mass (ASM) was measured using bioelectrical impedance analysis (BIA). Grip strength of the dominant hand and 4 m walking speed were additionally evaluated. Subjects were clinically classified as moderate sarcopenia according to appendicular skeletal muscle index 2 2 (ASMI; ASM/height2) <7.0 kg/m for men and <5.7 kg/m for women, as recommended by the Asian Working Group for Sarcopenia (AWGS) . In total, 50 sarcopenic and 46 normal subjects participated in the study. Blood samples were collected in serum separator tubes, frozen and stored until biomarker analysis. e I Th nstitutional Review Board of Seoul National University Hospital approved all procedures, and all meth- ods were performed in accordance with the relevant guidelines and regulations. All participants provided written informed consent upon enrollment. Enzyme-linked immunosorbent assay (ELISA). Serum levels of human IL-6, SPARC, MIF, and IGF-1 were measured using ELISA kits according to the manufacturer’s protocol (R&D systems, Inc., Minneapolis, MN, USA). Development of risk score. To develop a single risk score using all four biomarkers (IL-6, SPARC, MIF and 17,18 IGF-1), we adopted a previously developed strategy with regression analysis for multiple biomarkers . Briefly, serum levels were log transformed to reduce variations between the concentrations of each biomarker and used to generate logistic regression coefficients. The risk score for each individual was calculated as the sum of the risk score for each biomarker, which was derived by multiplying the serum level of a biomarker by its correspond- ing coefficient (Risk score = ∑ logistic regression coefficient of biomarker M × serum level of biomarker M ). i i Subjects were subsequently divided into two groups (high and low risk of sarcopenia) using the median cuto ff risk score as a threshold. Statistics. The significance of association between a risk group and clinically manifested sarcopenia was assessed using Fisher’s exact test. To estimate the utility of risk score as a diagnostic tool of sarcopenia, we cal- culated the area under curve (AUC) value using receiver operating characteristics (ROC) analysis. The signifi - cance of serum level differences between normal and sarcopenia groups was assessed using two-sample t-tests for each biomarker. Data were considered statistically significant at p-values < 0.05 and false discovery rate <0.3. All statistical analyses were performed using GraphPad Prism5 (GraphPad Software, Inc., USA) and R language environment (ver. 3.2.5). Results Baseline characteristics. Mean appendicular skeletal muscle index (ASMI) values of normal (46 sub- 2 2 2 jects) and sarcopenia (50 subjects) groups were 7.50 kg/m and 6.51 kg/m respectively in men, and 6.48 kg/m and 5.23 kg/m respectively in women. The sarcopenia group had weaker grip strength than the normal group (27.83 kgF vs 32.28 kgF in men and 17.06 kgF vs 20.86 kgF in women) but displayed no significant differences in gait speed. The baseline characteristics are shown in Table  1. Selection of biomarkers for diagnosis of sarcopenia. To identify effective molecular biomarkers for diagnosis of sarcopenia, candidates were selected by searching the relevant literature and subjected to immu- nochemical analyses using elderly people sera of normal and sarcopenia group that was clinically manifested based on AWGS. We examined 21 biomarker candidates, including proteins related to skeletal muscle function or metabolism and muscle-derived cytokines (myokines), angiotensin-converting enzyme (ACE) , insulin-like 20 21 growth factor 1 (IGF-1) , procollagen type III N-terminal peptide (P3NP) , fibroblast growth factor 21 22 23 24 25 (FGF21) , myostatin , growth differentiation factor 11 (GDF11) , meteorin-like (METRNL) , macrophage 26 27 28 migration inhibitory factor (MIF) , fatty acid binding protein 3 (FABP3) , ciliary neurotrophic factor (CNTF) , secreted protein acidic and rich in cysteine (SPARC) , brain-derived neurotrophic factor (BDNF), tumor necro- sis factor-α (TNF-α), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 15 (IL-15), interleukin 1β (IL-1β), monocyte chemotactic Protein 1 (MCP-1), transforming growth factor β 1 (TGFβ1), and vascular endothelial growth factor (VEGF) . Among the candidates investigated, serum levels of IL-6, SPARC, MIF and IGF-1 were significantly different between normal and sarcopenia groups (Fig. 1), but not the other biomarkers (data not shown). Serum lev- els of IL-6 were higher in the sarcopenia than the normal group (Fig. 1a; 1.64 ± 0.36 versus 0.86 ± 0.25 pg/ml, p = 0.015). Exceptionally, the IL-6 levels in three sarcopenia and six normal samples were undetectable with a commercially available ELISA kit that can detect concentrations as low as 0.1 pg/mL. Similarly, serum concen- trations of SPARC and MIF were higher in the sarcopenia than the normal group (Fig. 1b,c; 531.5 ± 40.65 versus 409.4 ± 35.47 pg/mL SPARC, p = 0.047, and 25.10 ± 1.19 versus 20.71 ± 0.89 ng/mL MIF, p = 0.008). Conversely, Scientific REPO R tS | (2018) 8:8574 | DOI:10.1038/s41598-018-26617-9 2 www.nature.com/scientificreports/ Men Women * * Normal Sarcopenia P-value Normal Sarcopenia P-value Number of subjects 18 18 28 32 ** Age (years) 76.00 ± 5.72 76.22 ± 5.62 0.907 71.86 ± 6.01 76.13 ± 6.05 0.008 ** Height (cm) 164.16 ± 5.02 161.56 ± 3.97 0.093 152.90 ± 5.27 147.14 ± 5.14 <0.001 ** Weight (kg) 65.68 ± 6.05 57.47 ± 6.29 <0.001 62.40 ± 6.58 49.85 ± 5.95 <0.001 ** Appendicular skeletal muscle mass (kg) 20.23 ± 2.09 16.97 ± 1.86 <0.001 15.23 ± 1.67 11.38 ± 1.29 <0.001 2 ** Appendicular skeletal muscle index (km/m ) 7.50 ± 0.41 6.51 ± 0.54 <0.001 6.48 ± 0.39 5.23 ± 0.30 <0.001 ** Grip strength (kgF) 32.28 ± 6.50 27.83 ± 5.09 0.029 20.86 ± 4.91 17.06 ± 2.71 <0.001 ** Gait speed (m/s) 0.99 ± 0.14 1.01 ± 0.16 0.685 1.00 ± 0.22 0.98 ± 0.15 0.741 Table 1. Baseline characteristics of the samples. *Independent t-test. **Mean ± standard deviation. Figure 1. Comparison of serum protein levels between normal and sarcopenic aged subjects. IL-6 (a), SPARC (b), MIF (c) and IGF-1 (d) protein levels in human serum were measured using sandwich ELISA. Box plots were used to visualize distribution of each serum protein level. P-values were obtained with two sample t-tests. serum IGF-1 levels were lower in the sarcopenia group (Fig. 1d; 58.16 ± 3.37 versus 72.61 ± 5.49, p = 0.039). Based on the collective results, IL-6, SPARC, MIF and IGF-1 were selected for analysis as potential biomarkers for sarcopenia. Application of risk score to predict sarcopenia. Combination of measurements of multiple biomarkers into a single score was required to integrate the collected information for easy diagnostic judgement. A risk score was generated by combination of the measurements of the four selected biomarkers (IL-6, SPARC, MIF and IGF-1) based on the logistic regression coefficient of each biomarker (Table  2). Using the median cutoff value of risk scores for all subjects (1.518), two groups with high-risk and low-risk scores were classified (Fig.  2a). The frequency of clinically identified sarcopenia was significantly higher in the high-risk than the low-risk group Scientific REPO R tS | (2018) 8:8574 | DOI:10.1038/s41598-018-26617-9 3 www.nature.com/scientificreports/ Protein Estimated β Std. Error P-value IL-6 0.151 0.062 0.016 SPARC 0.102 0.05 0.047 MIF 0.279 0.102 0.008 IGF-1 −0.129 0.062 0.041 Table 2. Univariate logistic regression analyses of 4 proteins. β indicates regression coefficient value. (Fig. 2b; p = 0.002, 95% confidence interval (CI) = 1.56–11.59), indicating that biomarker analysis is clinically valuable for sarcopenia diagnosis. When adapted to groups of men and women separately, biomarker analysis was successfully applied to identify sarcopenia in both genders, representing no gender differences in positive predictive value (p = 0.001, 95% CI = 2.28–171.69 in men; p = 0.009, 95% CI = 1.28–15.66 in women; Fig. 2c,d). However, the frequency of sarcopenia in the low-risk group was lower in men than women, signifying that the negative predictive value is higher in men. Significance of multiple biomarkers for diagnosis of sarcopenia. To establish whether the risk score based on multiple biomarkers is useful for diagnosis of sarcopenia, area under the ROC curve, sensitivity and specificity were assessed. When applying ROC analysis to risk score, significantly high AUC values were obtained for all population categories (AUC > 0.7). AUC was 0.763 in the overall elderly population and the best cuto ff value, defined as the point with highest multiplicity of sensitivity and specificity, was a risk score of 1.529 (Fig.  3a; 70% sensitivity and 78.26% specificity). AUC was 0.812 and the best cutoff value in elderly men (Fig.  3b; 88.88% sensitivity, 77.77% specificity) was a risk score of 1.543. In elderly women, AUC was 0.739 and a risk score of 1.505 (Fig. 3c; 68.75% sensitivity, 75% specificity) was the best cutoff value. To ascertain whether the optimal cutoff value based on ROC analysis improves predictability, subjects were dichotomized into two groups based on the above cutoff values of risk score. When applying the cutoff values of 1.529, 1.543, and 1.505 to the whole population, men and women groups, respectively, performance in classifying the high-risk sarcopenia group was improved (Supplementary Fig. 1). Our results collectively suggest that the risk score based on multiple biomark- ers selected in this study demonstrates predictive value for sarcopenia regardless of gender. Discussion While several candidate molecular biomarkers of sarcopenia have been proposed to date, none have been assessed for predictive ability. The four biomarkers analyzed in this study were not appropriate as single biomarkers for diagnosis of sarcopenia. Despite differences in serum levels between normal and sarcopenia groups with accept- able p-values (Fig. 1), none attained optimal cutoff values at which both sensitivity and specificity were > 70%, resulting in AUC <0.7 (data not shown). Interestingly, however, combination of the biomarker measurements into a single risk score enhanced the accuracy of diagnosis for sarcopenia. Sarcopenia group in our study involved elderly people with lower muscle mass (on the basis of AWGS), but excluded subjects with severe muscle weakness (grip strength <26 kgF for men and <18 kgF for women and gait speed <0.8 m/s) and potential accompanying diseases. This ‘moderate sarcopenia’ group showed slightly weaker muscle strength than the normal group but greater strength than the standard sarcopenia group (Table 1). er Th efore, selected multiple biomarkers in combination may be effectively utilized for early diagnosis of sarcope- nia before clinical manifestation of symptoms, such as weakness, disability, falls, loss of independence, and frailty. The above biomarkers are evidently correlated with skeletal muscle metabolism and function. IL-6, a well-known pro-inflammatory cytokine, was the first identified myokine . Earlier studies have reported that serum IL-6 is elevated chronically in cachexia and Duchenne muscular dystrophy (DMD) patients and acutely aer ex ft ercise, and decreases rapidly within an hour to pre-exercise levels . Serum IL-6 has been highlighted as a potential sarcopenia biomarker by several groups, but is yet to be statistically validated. SPARC, also known as osteonectin or basement-membrane protein 40, is an acidic extracellular matrix gly- 32–34 coprotein secreted by most tissues and involved in bone mineralization and cell-matrix interactions . SPARC is highly expressed in patients with various types of myopathy including DMD, inclusion body myositis, and congenital muscular dystrophy . Controversially, another study showed that SPARC overexpression extracellu- 36 37 larly inhibits muscle cell die ff rentiation , and its deficiency in mouse skeletal muscle causes myob fi er atrophy . Although reduced levels of SPARC in aged muscle may correlate with sarcopenia , changes in the circulating levels of SPARC with aging have not been documented. MIF, a pro-inflammatory cytokine, has been implicated in muscle damage, in view of its increased levels in 26 38 muscle of polymyositis patients . Since MIF is suggested to regulate glucose homeostasis and skeletal muscle is a major organ for glucose utilization, we speculate that circulating MIF levels may reflect the status of glucose metabolism in sarcopenia. IGF-1, also known as somatomedin C, is a well-known mediator of muscle growth and subsequent regenera- tion . IGF-1 signaling involving the activation of phosphatidylinositol-3-kinase and Akt (or protein kinase B) not only induces muscle hypertrophy via stimulation of protein synthesis pathways but also predominantly prevents activation of muscle atrophy pathways via inhibition of forkhead box O (FoxO) transcription factors . IGF-1 is produced by satellite cells in regenerating skeletal muscles and muscle-specific expression of the protein accel- erates regeneration of injured muscle by rapidly modulating the inflammatory response . Furthermore, IGF-1 triggers human myotube hypertrophy by accelerating the recruitment of reserve cells in human skeletal muscle . Scientific REPO R tS | (2018) 8:8574 | DOI:10.1038/s41598-018-26617-9 4 www.nature.com/scientificreports/ Figure 2. Risk score based on combination of four biomarkers (IL6, SPARC, MIF, and IGF-1) and frequency of sarcopenia. Bar graph of risk scores in 96 samples (a). Median risk score across samples was applied as a threshold for dividing into two risk groups (cutoff value = 1.518). Comparison of frequency of sarcopenia between low-risk and high-risk groups in whole population (b), Men (c), Women (d). P-values and confidence intervals (CI) were calculated with Fisher’s exact test. Figure 3. Significance of risk score based on multiple biomarkers for diagnosis of sarcopenia. Receiver operating characteristic (ROC) curves for each group. Whole population (a), Men (b), Women (c). The area under the ROC curve (AUC) was calculated for each group to determine the significance of multiple biomarkers in predicting sarcopenia. Effective multiple biomarkers for sarcopenia diagnosis are not confined to the four biomarkers assessed in this study. To reflect entire biological pathways associated with sarcopenia disease activity, optimal combinations of additional novel biomarkers require exploration. Furthermore, longitudinal studies are essential to determine whether analysis of multiple biomarkers can be employed for not only early diagnosis but also evaluating ther- apeutic interventions and prognosis prior to changes in muscle mass, strength or function. In addition, consid- ering that inflammation is a common mechanism in a variety of diseases associated with aging, such as cancer and diabetes in addition to sarcopenia, specific biomarkers should exclude the possibility of interference by other diseases . Despite the limitations of the current study, our results clearly support a new innovative approach in molecu- lar diagnosis and prognosis of sarcopenia using multiple biomarkers and provide a step in further understanding the mechanisms underlying sarcopenia and identifying novel therapeutic targets for improving health and quality of life in the elderly. References 1. Rosenberg, I. H. Sarcopenia: origins and clinical relevance. J Nutr 127, 990S–991S (1997). 2. Roubeno, R ff . Sarcopenia: a major modifiable cause of frailty in the elderly. J Nutr Health Aging 4, 140–142 (2000). 3. Cao, L. & Morley, J. E. 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Jacquemin, V., Furling, D., Bigot, A., Butler-Browne, G. S. & Mouly, V. IGF-1 induces human myotube hypertrophy by increasing cell recruitment. Exp Cell Res 299, 148–158, https://doi.org/10.1016/j.yexcr.2004.05.023 (2004). 44. Chung, H. Y. et al. Molecular inflammation: underpinnings of aging and age-related diseases. Ageing Res Rev 8, 18–30, https://doi. org/10.1016/j.arr.2008.07.002 (2009). Acknowledgements This study was supported by grants from the Bio and Medical Technology Development Program (2013M3A9B6076415, 2017M3A9D8048709 to K.-S.K.) of the National Research Foundation, which is funded by the Korean government (Ministry of Science, ICT and Future Planning), and the Korea Research Institute of Bioscience and Biotechnology Initiative Program. Scientific REPO R tS | (2018) 8:8574 | DOI:10.1038/s41598-018-26617-9 6 www.nature.com/scientificreports/ Author Contributions J.Y.K., H.H., J.Y.C., S.-M.L., H.B., E.-S.K., K.-P.L., S.G.C., and K.-S.K. designed the research strategy. J.Y.K. and H.H. performed experiments and S.-K.K. calculated risk score and conducted statistical analyses. J.Y.K., H.H., S.-K.K., S.G.C., and K.-S.K. wrote the manuscript and all authors analyzed and discussed the results and edited the manuscript. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-26617-9. Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 Scientific REPO R tS | (2018) 8:8574 | DOI:10.1038/s41598-018-26617-9 7

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