Associations of methyl donor and methylation inhibitor levels during anti-oxidant therapy in heart failure


Redox balance and methylation are crucial to homeostasis and are linked by the methionine-homocysteine cycle. We examined whether differences in methylation potential, measured as plasma levels of S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH), occur at baseline and during anti-oxidant therapy with the xanthine oxidase inhibitor allopurinol in patients with heart failure with reduced ejection fraction. We analyzed plasma samples collected at baseline and 24 weeks in the Xanthine Oxidase Inhibition for Hyperuricemic Heart Failure Patients (EXACT-HF) study, which randomized patients with heart failure with reduced ejection fraction to allopurinol or placebo. Associations between plasma levels of SAM, SAH, SAM/SAH ratio, and outcomes, including laboratory markers and clinical events, were assessed. Despite randomization, median SAM levels were significantly lower at baseline in the allopurinol group. SAH levels at 24 weeks, and change in SAM from baseline to week 24, were significantly higher in the group of patients randomized to allopurinol compared to the placebo group. A significant correlation was observed between change in SAH levels and change in plasma uric acid (baseline to 24-week changes) in the allopurinol group. There were no significant associations between levels of SAM, SAH, and SAM/SAH ratio and clinical outcomes. Our results demonstrate significant biological variability in SAM and SAH levels at baseline and during treatment with an anti-oxidant and suggest a potential mechanism for the lack of efficacy observed in trials of anti-oxidant therapy. These data also highlight the need to explore personalized therapy for heart failure.

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  1. 1.

    Bjelakovic G, Nikolova D, Gluud LL, Simonetti RG, Gluud C (2015) Antioxidant supplements for prevention of mortality in healthy participants and patients with various diseases. Sao Paulo Med J 133:164–165

    Article  Google Scholar 

  2. 2.

    Cyr AR, Domann FE (2011) The redox basis of epigenetic modifications: from mechanisms to functional consequences. Antioxid Redox Signal 15:551–589

    CAS  Article  Google Scholar 

  3. 3.

    Devi S, Kennedy RH, Joseph L, Shekhawat NS, Melchert RB, Joseph J (2006) Effect of longterm hyperhomocysteinemia on myocardial structure and function in hypertensive rats. Cardiovasc Pathol 15:75–82

    CAS  Article  Google Scholar 

  4. 4.

    Givertz MM, Mann DL, Lee KL, Ibarra JC, Velazquez EJ, Hernandez AF, Mascette AM, Braunwald E (2013) Xanthine oxidase inhibition for hyperuricemic heart failure patients: design and rationale of the EXACT-HF study. Circ Heart Fail 6:862–868

    Article  Google Scholar 

  5. 5.

    Givertz MM, Anstrom KJ, Redfield MM, Deswal A, Haddad H, Butler J, Tang WH, Dunlap ME, LeWinter M, Mann DL, Felker GM, O'Connor CM, Goldsmith SR, Ofili EO, Saltzberg MT, Margulies KB, Cappola TP, Konstam MA, Semigran MJ, McNulty S, Lee KL, Shah MR, Hernandez AF, NHLBI Heart Failure Clinical Research Network (2015) Effects of Xanthine Oxidase Inhibition in Hyperuricemic Heart Failure Patients: The Xanthine Oxidase Inhibition for Hyperuricemic Heart Failure Patients (EXACT-HF) Study. Circulation 131:1763–1771

    CAS  Article  Google Scholar 

  6. 6.

    Hoffman DR, Cornatzer WE, Duerre JA (1979) Relationship between tissue levels of Sadenosylmethionine, S-adenylhomocysteine, and transmethylation reactions. Can J Biochem 57:56–65

    CAS  Article  Google Scholar 

  7. 7.

    James SJ, Cutler P, Melnyk S, Jernigan S, Janak L, Gaylor DW, Neubrander JA (2004) Metabolic biomarkers of increased oxidative stress and impaired methylation capacity in children with autism. Am J Clin Nutr 80:1611–1617

    CAS  Article  Google Scholar 

  8. 8.

    Joseph J, Loscalzo J (2013) Methoxistasis: integrating the roles of homocysteine and folic acid in cardiovascular pathobiology. Nutrients 5:3235–3256

    CAS  Article  Google Scholar 

  9. 9.

    Joseph J, Loscalzo J (2013) Selenistasis: epistatic effects of selenium on cardiovascular phenotype. Nutrients 5:340–358

    CAS  Article  Google Scholar 

  10. 10.

    Joseph J, Joseph L, Devi S, Kennedy RH (2008) Effect of anti-oxidant treatment on hyperhomocysteinemia-induced myocardial fibrosis and diastolic dysfunction. J Heart Lung Transplant 27:1237–1241

    Article  Google Scholar 

  11. 11.

    Kerins DM, Koury MJ, Capdevila A, Rana S, Wagner C (2001) Plasma Sadenosylhomocysteine is a more sensitive indicator of cardiovascular disease than plasma homocysteine. Am J Clin Nutr 74:723–729

    CAS  Article  Google Scholar 

  12. 12.

    Klepacki J, Brunner N, Schmitz V, Klawitter J, Christians U, Klawitter J (2013) Development and validation of an LC-MS/MS assay for the quantification of the trans-methylation pathway intermediates S-adenosylmethionine and S-adenosylhomocysteine in human plasma. Clin Chim Acta 421:91–97

    CAS  Article  Google Scholar 

  13. 13.

    Labunskyy VM, Lee BC, Handy DE, Loscalzo J, Hatfield DL, Gladyshev VN (2011) Both maximal expression of selenoproteins and selenoprotein deficiency can promote development of type 2 diabetes-like phenotype in mice. Antioxid Redox Signal 14:2327–2336

    CAS  Article  Google Scholar 

  14. 14.

    Lippman SM, Klein EA, Goodman PJ, Lucia MS, Thompson IM, Ford LG, Parnes HL, Minasian LM, Gaziano JM, Hartline JA, Parsons JK, Bearden JD, Crawford ED, Goodman GE, Claudio J, Winquist E, Cook ED, Karp DD, Walther P, Lieber MM, Kristal AR, Darke AK, Arnold KB, Ganz PA, Santella RM, Albanes D, Taylor PR, Probstfield JL, Jagpal TJ, Crowley JJ, Meyskens FL, Baker LH, Coltman CA (2009) Effect of selenium and vitamin E on risk of prostate cancer and other cancers: the Selenium and Vitamin E Cancer Prevention Trial (SELECT). JAMA 301:39–51

    CAS  Article  Google Scholar 

  15. 15.

    Marti-Carvajal AJ, Sola I, Lathyris D, Dayer M (2017) Homocysteine-lowering interventions for preventing cardiovascular events. Cochrane Database Syst Rev 8:CD006612

    PubMed  Google Scholar 

  16. 16.

    Melnyk S, Pogribna M, Pogribny IP, Yi P, James SJ (2000) Measurement of plasma and intracellular S-adenosylmethionine and S-adenosylhomocysteine utilizing coulometric electrochemical detection: alterations with plasma homocysteine and pyridoxal 5'-phosphate concentrations. Clin Chem 46:265–272

    CAS  Article  Google Scholar 

  17. 17.

    Metes-Kosik N, Luptak I, Dibello PM et al (2012) Both selenium deficiency and modest selenium supplementation lead to myocardial fibrosis in mice via effects on redoxmethylation balance. Mol Nutr Food Res 56:1812–1824

    CAS  Article  Google Scholar 

  18. 18.

    Mosharov E, Cranford MR, Banerjee R (2000) The quantitatively important relationship between homocysteine metabolism and glutathione synthesis by the transsulfuration pathway and its regulation by redox changes. Biochemistry 39:13005–13011

    CAS  Article  Google Scholar 

  19. 19.

    Movassagh M, Choy MK, Knowles DA, Cordeddu L, Haider S, Down T, Siggens L, Vujic A, Simeoni I, Penkett C, Goddard M, Lio P, Bennett MR, Foo RSY (2011) Distinct epigenomic features in end-stage failing human hearts. Circulation 124:2411–2422

    Article  Google Scholar 

  20. 20.

    Niedzwiecki MM, Hall MN, Liu X, Oka J, Harper KN, Slavkovich V, Ilievski V, Levy D, van Geen A, Mey JL, Alam S, Siddique AB, Parvez F, Graziano JH, Gamble MV (2013) Blood glutathione redox status and global methylation of peripheral blood mononuclear cell DNA in Bangladeshi adults. Epigenetics 8:730–738

    CAS  Article  Google Scholar 

  21. 21.

    Pajares MA, Duran C, Corrales F, Pliego MM, Mato JM (1992) Modulation of rat liver Sadenosylmethionine synthetase activity by glutathione. J Biol Chem 267:17598–17605

    CAS  Article  Google Scholar 

  22. 22.

    Rafeq Z, Roh JD, Guarino P, Kaufman J, Joseph J (2013) Adverse myocardial effects of Bvitamin therapy in subjects with chronic kidney disease and hyperhomocysteinaemia. Nutr Metab Cardiovasc Dis 23:836–842

    CAS  Article  Google Scholar 

  23. 23.

    Rajasekaran NS, Connell P, Christians ES, Yan LJ, Taylor RP, Orosz A, Zhang XQ, Stevenson TJ, Peshock RM, Leopold JA, Barry WH, Loscalzo J, Odelberg SJ, Benjamin IJ (2007) Human alpha B-crystallin mutation causes oxido-reductive stress and protein aggregation cardiomyopathy in mice. Cell 130:427–439

    CAS  Article  Google Scholar 

  24. 24.

    Stranges S, Marshall JR, Natarajan R, Donahue RP, Trevisan M, Combs GF, Cappuccio FP, Ceriello A, Reid ME (2007) Effects of long-term selenium supplementation on the incidence of type 2 diabetes: a randomized trial. Ann Intern Med 147:217–223

    Article  Google Scholar 

  25. 25.

    Sun B, Cheng L, Xiong Y et al (2017) PSORS1C1 Hypomethylation Is Associated with Allopurinol-Induced Severe Cutaneous Adverse Reactions during Disease Onset Period: A Multicenter Retrospective Case-Control Clinical Study in Han Chinese. Front Pharmacol 8:923

    Article  Google Scholar 

  26. 26.

    Tsutsui H, Kinugawa S, Matsushima S (2011) Oxidative stress and heart failure. Am J Physiol Heart Circ Physiol 301:H2181–H2190

    CAS  Article  Google Scholar 

  27. 27.

    Vasan RS, Beiser A, D'Agostino RB, Levy D, Selhub J, Jacques PF, Rosenberg IH, Wilson PW (2003) Plasma homocysteine and risk for congestive heart failure in adults without prior myocardial infarction. JAMA 289:1251–1257

    CAS  Article  Google Scholar 

  28. 28.

    Vivekananthan DP, Penn MS, Sapp SK, Hsu A, Topol EJ (2003) Use of antioxidant vitamins for the prevention of cardiovascular disease: meta-analysis of randomised trials. Lancet 361:2017–2023

    CAS  Article  Google Scholar 

  29. 29.

    Wang X, Cui L, Joseph J, Jiang B, Pimental D, Handy DE, Liao R, Loscalzo J (2012) Homocysteine induces cardiomyocyte dysfunction and apoptosis through p38 MAPK-mediated increase in oxidant stress. J Mol Cell Cardiol 52:753–760

    CAS  Article  Google Scholar 

  30. 30.

    Wei H, Liang F, Meng G, Nie Z, Zhou R, Cheng W, Wu X, Feng Y, Wang Y (2016) Redox/methylation mediated abnormal DNA methylation as regulators of ambient fine particulate matter-induced neurodevelopment related impairment in human neuronal cells. Sci Rep 6:33402

    CAS  Article  Google Scholar 

  31. 31.

    Xiao Y, Zhang Y, Wang M, Li X, Su D, Qiu J, Li D, Yang Y, Xia M, Ling W (2013) Plasma S-adenosylhomocysteine is associated with the risk of cardiovascular events in patients undergoing coronary angiography: a cohort study. Am J Clin Nutr 98:1162–1169

    CAS  Article  Google Scholar 

  32. 32.

    Yi P, Melnyk S, Pogribna M, Pogribny IP, Hine RJ, James SJ (2000) Increase in plasma homocysteine associated with parallel increases in plasma S-adenosylhomocysteine and lymphocyte DNA hypomethylation. J Biol Chem 275:29318–29323

    CAS  Article  Google Scholar 

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The authors gratefully acknowledge the EXACT-HF study investigators, coordinators, and patients for their time and effort on behalf of the clinical trial. We also thank the staff of the core biomarker laboratory at the University of Vermont, under the direction of Russel Tracy PhD, for their handling of clinical specimens. The authors would also like to acknowledge the Metabolomics Shared Resource at Georgetown University (Washington, DC, USA) that is partially supported by NIH/NCI/CCSG grant P30-CA051008.


This work was supported by grants from the National Institutes of Health: NHLBI coordinating center: U10HL084904; and regional clinical center: U10HL110337

Author information




Drs. Joseph and Givertz secured funding, devised and oversaw the project, interpreted data, and wrote and revised the manuscript. Ms. Giczewska and Dr. Alhanti analyzed the data, wrote the “Methods” section of the manuscript, and reviewed the manuscript. Dr. Cheema supervised the methylation marker analysis, interpreted results, and provided comments on the manuscript. Drs. Handy, Mann, and Loscalzo provided input on the interpretation of data and provided comments on the manuscript.

Corresponding author

Correspondence to Jacob Joseph.

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Ethics approval and consent to participate

This is a sub-study utilizing data and blood samples collected as part of the primary study. No additional data or blood collection was performed as part of this study. This sub-study was approved by Institutional Review Boards of Brigham and Women’s Hospital and Duke University Medical Center. Informed consent was obtained from participants as part of the main study EXACT-HF.

Competing interests

The authors declare no competing interests.

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Key Points:

• Redox balance and methylation reactions are linked through the methionine-homocysteine cycle.

• The body’s methylation potential varies widely among individuals with heart failure.

• Methylation potential is altered by anti-oxidant therapy.

• These findings may explain the neutral results of clinical trials of anti-oxidant therapy.

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Joseph, J., Giczewska, A., Alhanti, B. et al. Associations of methyl donor and methylation inhibitor levels during anti-oxidant therapy in heart failure. J Physiol Biochem (2021).

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  • Methylation
  • Redox balance
  • Anti-oxidant therapy
  • Heart failure