NMR-based plasma metabolic profiling in patients with unstable angina

Document Type : Original Article


1 Department of Genetics & Molecular Medicine, Faculty of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran

2 Chemistry Group, Faculty of Basic Sciences, University of MohagheghArdabili, Ardabil, Iran

3 Department of Cardiology, Mousavi Hospital, Zanjan University of Medical Sciences, Zanjan, Iran

4 Zanjan Metabolic Diseases Research Center, Zanjan University of Medical Sciences, Zanjan, Iran

5 Cardiac Surgery Department, Rohani Hospital, Babol University of Medical Sciences, Babol, Iran

6 International Agency for Research on Cancer,150cours Albert Thomas, 69372 Lyon CEDEX 08, Lyon, France

7 Cancer Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan, Iran


Objective(s): Unstable angina (UA) is a form of the acute coronary syndrome (ACS) that affects more than a third of the population before age 70. Due to the limitations of diagnostic tests, appropriate identification of UA is difficult. In this study, we proceeded to investigate metabolite profiling in UA patients compared with controls to determine potential candidate biomarkers.
Materials and Methods: Ninety-four plasma samples from UA and 32 samples from controls were analyzed based on 1H NMR spectroscopy. The raw data were processed, analyzed, and subjected to partial least squares-discrimination analysis (PLS-DA), a supervised classification method with a good separation of control and UA patients was observed. The most important variables (VIP) ≥1 were selected and submitted to MetaboAnalyst pathway enrichment to identify the most important ones.
Results: We identified 17 disturbed metabolites in UA patients in comparison with the controls.   These metabolites are involved in various biochemical pathways such as steroid hormone biosynthesis, aminoacyl-tRNA biosynthesis, and lysine degradation. Some of the metabolites were deoxycorticosterone, 17-hydroxyprogesterone, androstenedione, androstanedione, etiocholanolone, estradiol, 2-hydroxyestradiol, 2-hydroxyestrone, 2-methoxyestradiol, and 2-methoxyestrone. In order to determine test applicability in diagnosing UA, a diagnostic model was further created using the receiver operator characteristic (ROC) curve. The areas under the curve (AUC), sensitivity, specificity, and precision were 0.87, 90%, 65%, and 91%, respectively, for diagnosing of UA.
Conclusion: These metabolites could not only be useful for the diagnosis of UA patients but also provide more information for further deciphering of the biological processes of UA.


1. Amsterdam EA, Wenger NK, Brindis RG, Casey DE, Jr., Ganiats TG, Holmes DR, Jr., et al. 2014 AHA/ACC guideline for the management of patients with non-ST-elevation acute coronary syndromes: executive summary: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. Circulation 2014; 130:2354-2394.
2. Kibos A, Guerchicoff A. Susceptibility genes for coronary heart disease and myocardial infarction. Acute Card Care 2011; 13:136-142.
3. Ambrose JA, Winters SL, Stern A, Eng A, Teichholz LE, Gorlin R, et al. Angiographic morphology and the pathogenesis of unstable angina pectoris. J Am Coll Cardiol 1985; 5:609-616.
4. Allender S, Scarborough P, O’Flaherty M, Capewell S. 20th century CHD morality in England and Wales: population trends in CHD risk factors and coronary death. BMC Public Health 2008; 8:148.
5. Unal B, Critchley JA, Capewell S. Explaining the decline in coronary heart disease mortality in England and Wales between 1981 and 2000. Circulation 2004; 109:1101-1107.
6. Das R, Kilcullen N, Morrell C, Robinson MB, Barth JH, Hall AS. The British Cardiac Society Working Group definition of myocardial infarction: implications for practice. Heart 2006; 92:21-26.
7. Ali SE, Farag MA, Holvoet P, Hanafi RS, Gad MZ. A comparative metabolomics approach reveals early biomarkers for metabolic response to acute myocardial infarction. Sci Rep 2016; 6:36359.
8. Libby P, Tabas I, Fredman G, Fisher EA. Inflammation and its resolution as determinants of acute coronary syndromes. Circ Res 2014; 114:1867-1879.
9. Barderas MG, Laborde CM, Posada M, de la Cuesta F, Zubiri I, Vivanco F, et al. Metabolomic profiling for identification of novel potential biomarkers in cardiovascular diseases. Biomed Res Int 2011; 2011.
10. Amsterdam EA, Wenger NK, Brindis RG, Casey DE, Jr., Ganiats TG, Holmes DR, Jr., et al. 2014 AHA/ACC Guideline for the Management of Patients with Non-ST-Elevation Acute Coronary Syndromes: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol 2014; 64:e139-e228.
11. Ritchie JL, Bateman TM, Bonow RO, Crawford MH, Gibbons RJ, Hall RJ, et al. Guidelines for clinical use of cardiac radionuclide imaging: a report of the American College of Cardiology/American Heart Association Task Force on assessment of diagnostic and therapeutic cardiovascular procedures (Committee on Radionuclide Imaging)—developed in collaboration with the American Society of Nuclear Cardiology. J Nucl Cardiol 1995; 2:172-192.
12. Sabatine MS, Liu E, Morrow DA, Heller E, McCarroll R, Wiegand R, et al. Metabolomic identification of novel biomarkers of myocardial ischemia. Circulation 2005; 112:3868-3875.
13. Robert P. Giugliano CPC, and Eugene Braunwald. Non–ST Elevation Acute Coronary Syndromes. Braunwald’s Heart Disease, 10th Edition 2015:1338.
14. Stringer KA, McKay RT, Karnovsky A, Quemerais B, Lacy P. Metabolomics and Its Application to Acute Lung Diseases. Front Immunol 2016; 7:44.
15. Gerszten RE, Wang TJ. The search for new cardiovascular biomarkers. Nature 2008; 451:949-952.
16. Pouralijan Amiri M, Khoshkam M, Salek RM, Madadi R, Faghanzadeh Ganji G, Ramazani A. Metabolomics in early detection and prognosis of acute coronary syndrome. Clin Chim Acta 2019; 495:43-53.
17. Patti GJ, Yanes O, Siuzdak G. Innovation: Metabolomics: the apogee of the omics trilogy. Nat Rev Mol Cell Biol 2012; 13:263-269.
18. Capati A, Ijare OB, Bezabeh T. Diagnostic Applications of Nuclear Magnetic Resonance-Based Urinary Metabolomics. Magn Reson Insights 2017; 10:1178623x17694346.
19. Schoeman J, Loots D. Improved disease characterisation and diagnostics using metabolomics: A review. Cell Tissue Res 2011; 11:2673.
20. Emwas AH, Salek R, L. Griffin J, Merzaban J. NMR-based metabolomics in human disease diagnosis: Applications, limitations, and recommendations. Metabolomics 2013; 9:1048-1072.
21. Lewis GD, Asnani A, Gerszten RE. Application of metabolomics to cardiovascular biomarker and pathway discovery. J Am Coll Cardiol 2008; 52:117-123.
22. Li Z, Liu X, Wang J, Gao J, Guo S, Gao K, et al. Analysis of urinary metabolomic profiling for unstable angina pectoris disease based on nuclear magnetic resonance spectroscopy. Mol Biosyst 2015; 11:3387-3396.
23. Sun M, Gao X, Zhang D, Ke C, Hou Y, Fan L, et al. Identification of biomarkers for unstable angina by plasma metabolomic profiling. Mol Biosyst 2013; 9:3059-3067.
24. Shi C-H, Zhao H-H, Hou N, Chen J-X, Shi Q, Xu X-G, et al. Identifying metabolite and protein biomarkers in Unstable angina in-patients by feature selection based data mining method. Chem Res Chinese U 2011; 27:87-93.
25. Yao W, Gao Y, Wan Z. Serum metabolomics profiling to identify biomarkers for unstable angina. Biomed Res Int 2017; 2017.
26. Zhao H, Chen J, Shi Q, Ma X, Yang Y, Luo L, et al. Metabolomics-based study of clinical and animal plasma samples in coronary heart disease with blood stasis syndrome. Evid Based Complement Alternat Med 2012.
27. Loria JP, Rance M, Palmer AG. A relaxation-compensated Carr− Purcell− Meiboom− Gill sequence for characterizing chemical exchange by NMR spectroscopy. J Am Chem Soc 1999; 121:2331-2332.
28. van den Berg RA, Hoefsloot HC, Westerhuis JA, Smilde AK, van der Werf MJ. Centering, scaling, and transformations: improving the biological information content of metabolomics data. BMC Genomics 2006; 7:142.
29. Eick K, Pohnert G. Simplifying complexity in metabolomics. Chem Biol 2015; 22:567-568.
30. Wishart DS, Tzur D, Knox C, Eisner R, Guo AC, Young N, et al. HMDB: the Human Metabolome Database. Nucleic Acids Res 2007; 35:D521-526.
31. Chong J, Soufan O, Li C, Caraus I, Li S, Bourque G, et al. MetaboAnalyst 4.0: towards more transparent and integrative metabolomics analysis. Nucleic Acids Res 2018; 46:W486-W494.
32. Newton R, Holden NS. Separating transrepression and transactivation: a distressing divorce for the glucocorticoid receptor? Mol Pharmacol 2007; 72:799-809.
33.  Stier CT, Jr., Chander PN, Rocha R. Aldosterone as a mediator in cardiovascular injury. Cardiol Rev 2002; 10:97-107.
34. Buonafine M, Bonnard B, Jaisser F. Mineralocorticoid receptor and cardiovascular disease. Am J Hypertens 2018; 31:1165-1174.
35. Kuster GM, Kotlyar E, Rude MK, Siwik DA, Liao R, Colucci WS, et al. Mineralocorticoid receptor inhibition ameliorates the transition to myocardial failure and decreases oxidative stress and inflammation in mice with chronic pressure overload. Circulation 2005; 111:420-427.
36. Marzolla V, Armani A, Mammi C, Moss ME, Pagliarini V, Pontecorvo L, et al. Essential role of ICAM-1 in aldosterone-induced atherosclerosis. Int J Cardiol 2017; 232:233-242.
37. Sartorio CL, Fraccarollo D, Galuppo P, Leutke M, Ertl G, Stefanon I, et al. Mineralocorticoid receptor blockade improves vasomotor dysfunction and vascular oxidative stress early after myocardial infarction. Hypertension 2007; 50:919-925.
38. Villablanca AC, Jayachandran M, Banka C. Atherosclerosis and sex hormones: current concepts. Clin Sci (Lond) 2010; 119:493-513.
39. Schoenhagen P, Nissen SE, Tuzcu EM. Coronary arterial remodeling: from bench to bedside. Curr Atheroscler Rep 2003; 5:150-154.
40. Yeap BB. Sex steroids and cardiovascular disease. Asian J Androl 2014; 16:239-247.
41. Mirando AC, Francklyn CS, Lounsbury KM. Regulation of angiogenesis by aminoacyl-tRNA synthetases. Int J Mol Sci 2014; 15:23725-23748.
42. Yao P, Fox PL. Aminoacyl-tRNA synthetases in medicine and disease. EMBO Mol Med 2013; 5:332-343.
43. Cylwik D, Mogielnicki A, Buczko W. L-arginine and cardiovascular system. Pharmacol Rep 2005; 57:14-22.
44. Tousoulis D, Böger RH, Antoniades C, Siasos G, Stefanadi E, Stefanadis C. Mechanisms of Disease: L-arginine in coronary atherosclerosis—a clinical perspective. Nat Clin Pract Card 2007; 4:274-283.
45. Tentolouris C, Tousoulis D, Goumas G, Stefanadis C, Davies G, Toutouzas P. L-Arginine in coronary atherosclerosis. Int J Cardiol 2000; 75:123-128.
46. Selhub J, Troen AM. Sulfur amino acids and atherosclerosis: a role for excess dietary methionine. Ann N Y Acad Sci 2016; 1363:18-25.
47. Virtanen JK, Voutilainen S, Rissanen TH, Happonen P, Mursu J, Laukkanen JA, et al. High dietary methionine intake increases the risk of acute coronary events in middle-aged men. Nutr Metab Cardiovasc Dis 2006; 16:113-120.
48. Garlick PJ. Toxicity of methionine in humans. J Nutr 2006; 136:1722s-1725s.
49. Ganguly P, Alam SF. Role of homocysteine in the development of cardiovascular disease. Nutr J 2015; 14:6.
50. Vécsei L, Szalárdy L, Fülöp F, Toldi J. Kynurenines in the CNS: recent advances and new questions. Nat Rev Drug Discov 2012; 12:64.
51. Liu G, Chen S, Zhong J, Teng K, Yin Y. Crosstalk between tryptophan metabolism and cardiovascular disease, mechanisms, and therapeutic implications. Oxid Med Cell Longev 2017; 2017:1602074.
52. Mangge H, Summers KL, Meinitzer A, Zelzer S, Almer G, Prassl R, et al. Obesity-related dysregulation of the tryptophan-kynurenine metabolism: role of age and parameters of the metabolic syndrome. Obesity (Silver Spring) 2014; 22:195-201.
53. Mangge H, Stelzer I, Reininghaus EZ, Weghuber D, Postolache TT, Fuchs D. Disturbed tryptophan metabolism in cardiovascular disease. Curr Med Chem 2014; 21:1931-1937.
54. Niinisalo P, Oksala N, Levula M, Pelto-Huikko M, Jarvinen O, Salenius JP, et al. Activation of indoleamine 2,3-dioxygenase-induced tryptophan degradation in advanced atherosclerotic plaques: Tampere vascular study. Ann Med 2010; 42:55-63.
55. Wirleitner B, Rudzite V, Neurauter G, Murr C, Kalnins U, Erglis A, et al. Immune activation and degradation of tryptophan in coronary heart disease. Eur J Clin Invest 2003; 33:550-554.
56. Murr C, Grammer TB, Meinitzer A, Kleber ME, Marz W, Fuchs D. Immune activation and inflammation in patients with cardiovascular disease are associated with higher phenylalanine to tyrosine ratios: the ludwigshafen risk and cardiovascular health study. J Amino Acids 2014; 2014:783730.
57. Neurauter G, Schrocksnadel K, Scholl-Burgi S, Sperner-Unterweger B, Schubert C, Ledochowski M, et al. Chronic immune stimulation correlates with reduced phenylalanine turnover. Curr Drug Metab 2008; 9:622-627.
58. Capuron L, Schroecksnadel S, Feart C, Aubert A, Higueret D, Barberger-Gateau P, et al. Chronic low-grade inflammation in elderly persons is associated with altered tryptophan and tyrosine metabolism: role in neuropsychiatric symptoms. Biol Psychiatry 2011; 70:175-182.
59. Wu HQ, Ungerstedt U, Schwarcz R. L-alpha-aminoadipic acid as a regulator of kynurenic acid production in the hippocampus: a microdialysis study in freely moving rats. Eur J Pharmacol 1995; 281:55-61.
60. Lin H, Levison BS, Buffa JA, Huang Y, Fu X, Wang Z, et al. Myeloperoxidase-mediated protein lysine oxidation generates 2-aminoadipic acid and lysine nitrile in vivo. Free Radic Biol Med 2017; 104:20-31.
61. Zeitoun-Ghandour S, Leszczyszyn OI, Blindauer CA, Geier FM, Bundy JG, Sturzenbaum SR. C. elegans metallothioneins: response to and defence against ROS toxicity. Mol Biosyst 2011; 7:2397-2406.
62. Jung K, Reszka R, Kamlage B, Bethan B, Stephan C, Lein M, et al. Tissue metabolite profiling identifies differentiating and prognostic biomarkers for prostate carcinoma. Int J Cancer 2013; 133:2914-2924.
63. Mihalik SJ, Moser HW, Watkins PA, Danks DM, Poulos A, Rhead WJ. Peroxisomal L-pipecolic acid oxidation is deficient in liver from Zellweger syndrome patients. Pediatr Res 1989; 25:548-552.
64. Armstrong DW, Zukowski J, Ercal N, Gasper M. Stereochemistry of pipecolic acid found in the urine and plasma of subjects with peroxisomal deficiencies. J Pharm Biomed Anal 1993; 11:881-886.
65. Langeland R. [Construction of facilities in the health sector]. Sykepleien 1976; 63:782-784, 793.