Non-invasive Sampling for Assessment of Oxidative Stress and Pro-inflammatory Cytokine Levels in Beta-Thalassaemia Major Patients

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Background: Beta-thalassaemia (β-thalassaemia) major patients are severely anaemic and require life-long blood transfusions for survival. These patients require iron-chelation therapy as a result of iron overload due to the monthly blood transfusions. The iron over load can cause oxidative damage and pro-inflammation and therefore, hasten mortality. Thus, regular monitoring of the oxidative stress and pro-inflammation status may be useful in these patients.

Methods: Measurement of biomarkers is usually performed on serum samples but the evaluation in non-invasive samples such as saliva would be more favourable in paediatric cases. In this study, the levels of pro-inflammatory cytokines such as tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) as well as oxidative indices such as lipid hydro peroxide, advanced oxidation protein products (AOPP), ferric reducing antioxidant power (FRAP), uric acid (UA) and glutathione peroxidase (GPx) activity in a total of 65 β-thalassaemia major patients (all on iron chelation) and 55 healthy control subjects were assessed. All the above biochemical parameters, measured using well established assay techniques, were detectable in saliva samples.

Results: Non parametric analyses showed that lipid hydroperoxide (LH) and glutathione peroxidase (GPx) activities were significantly higher in β-thalassaemia major patients. All other parameters were not significantly different between patient and control groups implying that iron chelation therapy was successful in attenuating oxidative stress. Strong positive correlation was observed between FRAP and UA levels. There was also a notable difference in tumour necrosis factor-α (TNF-α) between the patients and healthy controls when analysed according to ethnicity and age. AOPP level in β+-thalassaemia homozygous patients were significantly higher than β+/β0-compound heterozygous and β0-thalassaemia homozygous patients.

Conclusion: Saliva may serve as a reliable, non-invasive sample which can be used to assess oxidative indices and pro-inflammatory cytokines in β-thalassaemia major patients.

1. George E, Tan JAMA. Genotype-phenotype diversity of beta-thalassemia in Malaysia: treatment options and emerging therapies. Med J Malaysia. 2010; 65(4):256-60.

2. Abdul WJ, Naznin M, Nora MZ, Suzanah AR, Zulaiha M, Aidil Faszrul AR, Kamaruzaman WS. Thalassaemia: a study on the perception of patients and family members. Med J Malaysia. 2011;6(4):326-34.

3. Bank A. Understanding globin regulation in β-thalassemia: it’s as simple as α, β, γ, δ. J Clin Invest. 2005 Jun 1; 115(6): 1470–3. DOI: 10.1172/JCI25398

4. Liu S, McConnell SC, Ryan TM. Erythropoiesis in the Absence of Adult Hemoglobin. Mol Cell Biol. 2013 Jun;33(11):2241-51. DOI: 10.1128/MCB.01734-12

5. Cappellini MD, Cohen A, Eleftheriou A, et al. Guidelines for the Clinical Management of Thalassaemia [Internet]. 2nd Revised edition. Nicosia (CY): Thalassaemia International Federation; 2008. Chapter 2, Blood Transfusion Therapy in β-Thalassaemia Major.

6. Kuppusamy UR, Tan JAMA. Chelation therapy with Desferrioxamine does not normalize ferritin level but attenuates oxidative damage and improves total antioxidant level in Malaysian Chinese β-thalassaemia major patients. West Indian Med J. 2011;60(1):3-8.

7. Walter PB, Macklin EA, Porter J, Evans P, Kwiatkowski JL, Neufeld EJ, Coates T, Gardina PJ, Vichinsky E, Olivieri N, Alberti D, Holland J, Harmatz, P. Inflammation and oxidant-stress in β-thalassemia patients treated with iron chelators deferasirox (ICL670) or deferoxamine: an ancillary study of the Novartis CICL670A0107 trial. Haematologica. 2008;93(6):817-25. DOI: 10.3324/haematol.11755

8. Metwalley KA, El-Saied ARAH. Glucose homeostasis in Egyptian children and adolescents with β-thalassemia major: relationship to oxidative stress. Indian J Endocrinol Metab. 2014;8(3):333-9. DOI: 10.4103/2230-8210.131169

9. Rayegani SM, Azadi A. Lateral antebrachial cutaneous nerve injury induced by phlebotomy. J Brachial Plex Peripher Nerve Inj. 2007; 2:6-8.

10. Hegde MN, Hegde ND, Ashok A, Shetty S. Evaluation of total antioxidant capacity of saliva and serum in caries-free and caries-active adults: an in-vivo study. Indian J Dent Res. 2013;24:164-7. DOI: 10.4103/0970-9290.116670

11. Shetty SR, Babu S, Kumari S, Shetty P, Hegde S, Castelino R. Status of salivary lipid peroxidation in oral cancer and precancer. Indian J Med Paediatr Oncol. 2014;35(2):156–8. DOI: 10.4103/0971-5851.138990

12. Soukup M, Biesiada I, Henderson A, Idowu B, Rodeback D, Ridpath L, et al. Salivary uric acid as a non-invasive biomarker of metabolic syndrome. Diabetol Metab Syndr. 2012;4(1):14. DOI: 10.1186/1758-5996-4-14

13. Sculley DV, Evans SCL. Salivary antioxidants and periodontal disease status. Proc Nutr Soc. 2002;61:137-43. DOI: 10.1079/PNS2001141

14. Suh KI, Kim YK, Kho SH. Salivary levels of IL-1β, IL-6, IL-8 and TNF-α in patients with burning mouth syndrome. Arch Oral Biol. 2009;54:797-802. DOI: 10.1016/j.archoralbio.2009.05.007

15. Wolfram R, Oguogho A, Palumbo B, Sinzinger H. Enhanced oxidative stress in coronary heart disease and chronic heart failure as indicated by an increased 8-epi-PGF2α. Eur J Heart Fail. 2005;7:167–72. DOI: 10.1016/j.ejheart.2004.05.007

16. Olivieri NF, Pakbaz Z, Vichinsky E. Hb E/beta-thalassaemia: a common & clinically diverse disorder. The Indian Journal of Medical Research. 2011;134(4):522-31.

17. Devaux M. In: Exploring the Relationship between Education and Obesity, OECD. Economic Studies. 2011;1.

18. Sudan R, Bhagat M, Gupta S, Singh J, Koul A. Iron (FeII) Chelation, Ferric Reducing Antioxidant Power, and Immune Modulating Potential of Arisaema jacquemontii (Himalayan Cobra Lily). BioMed Res Int. 2014;2014, Article ID 179865, 7 pages.

19. Marsche G, Frank S, Hrzenjak A, Holzer M, Dirnberger S, Wadsack C, et al. Plasma-Advanced Oxidation Protein Products Are Potent High-Density Lipoprotein Receptor Antagonists In Vivo. Circ Res. 2009 Mar 27;104(6):750-7. DOI: 10.1161/CIRCRESAHA.108.193169

20. Siddique YH, Ara G, Afzal M. Estimation of Lipid Peroxidation Induced by Hydrogen Peroxide in Cultured Human Lymphocytes. Dose-Response. 2012; 10(1):1-10. DOI: 10.2203/dose-response.10-002.Siddique

21. Rahman K. Studies on free radicals, antioxidants, and co-factors. Clinical Interventions in Aging. 2007;2(2):219-36.

22. POWERS SK, JACKSON MJ. Exercise-Induced Oxidative Stress: Cellular Mechanisms and Impact on Muscle Force Production. Physiol Rev. 2008 Oct; 88(4): 1243–76. DOI: 10.1152/physrev.00031.2007

23. Duplancic D, Kukoc-Modun L, Modun D, Radic N. Simple and Rapid Method for the Determination of Uric Acid-Independent Antioxidant Capacity. Molecules. 2011 Aug 17;16(8):7058-68. DOI: 10.3390/molecules16087058

24. Fabbrini E, Serafini M, Colic Baric I, Hazen SL, Klein S. Effect of Plasma Uric Acid on Antioxidant Capacity, Oxidative Stress, and Insulin Sensitivity in Obese Subjects. Diabetes. 2014;63(3):976-81. DOI: 10.2337/db13-1396

25. Taylor EL, Armstrong KR, Perrett D, Hattersley AT, Winyard PG. Optimisation of an Advanced Oxidation Protein Products Assay: Its Application to Studies of Oxidative Stress in Diabetes Mellitus. Oxidative Medicine and Cellular Longevity. 2015;2015, Article ID 496271, 10 pages.

26. Daiber A, Daub S, Bachschmid M, Schildknecht S, Oelze M, Steven S, et al. Protein Tyrosine Nitration and Thiol Oxidation by Peroxynitrite—Strategies to Prevent These Oxidative Modifications. Int J Mol Sci. 2013 Apr 8;14(4):7542-70. DOI: 10.3390/ijms14047542

27. Fisher-Wellman K, Bloomer RJ. Acute exercise and oxidative stress: a 30 year history. Dyn Med. 2009 Jan 13;8:1. DOI: 10.1186/1476-5918-8-1

28. Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants and functional foods: Impact on human health. Pharmacogn Rev. 2010 Jul-Dec; 4(8): 118–126. DOI: 10.4103/0973-7847.70902

29. Tuttolomondo A, Pecoraro R, Pinto A. Studies of selective TNF inhibitors in the treatment of brain injury from stroke and trauma: a review of the evidence to date. Drug Des Devel Ther. 2014 Nov 7;8:2221-38. DOI: 10.2147/DDDT.S67655

30. Ajuwon OR, Oguntibeju OO, Marnewick JL. Amelioration of lipopolysaccharide-induced liver injury by aqueous rooibos (Aspalathus linearis) extract via inhibition of pro-inflammatory cytokines and oxidative stress. BMC Complementary and Alternative Medicine 2014;14:392. DOI: 10.1186/1472-6882-14-392

31. de Cássia da Silveira e Sá R, Andrade LN, de Sousa DP. A Review on Anti-Inflammatory Activity of Monoterpenes. Molecules. 2013;18:1227-54. DOI: 10.3390/molecules18011227

32. Puebla-Osorio N, Damiani E, Bover L, Ullrich SE. Platelet-activating factor induces cell cycle arrest and disrupts the DNA damage response in mast cells. Cell Death Dis. 2015 May 7;6:e1745.

33. Fisher G, Alvarez JA, Ellis AC, Granger WM, Ovalle F, Man CD, et al. Race differences in the association of oxidative stress with insulin in African- and European-American women. Obesity (Silver Spring). 2012 May;20(5):972-7. DOI: 10.1038/oby.2011.355

34. Kuppusamy UR, Indran M, Rokiah P. Glycaemic control in relation to xanthine oxidase and antioxidant indices in Malaysian type 2 diabetes patients. Diabet Med. 2005 Oct;22(10):1343-6. DOI: 10.1111/j.1464-5491.2005.01630.x

35. Flora SJS, Pachauri V. Chelation in Metal Intoxication. Int J Environ Res Public Health. 2010 Jul;7(7):2745-88. DOI: 10.3390/ijerph7072745

36. Chinelato, IS, Carrocini, GCdS, & Bonini-Domingos, CR. (2011). XmnI polymorphism frequency in heterozygote beta thalassemia subjects and its relation to fetal hemoglobin levels. Rev Bras Hematol Hemoter. 2011; 33(6):483. DOI: 10.5581/1516-8484.20110128

37. Manu Rathee, Mohaneesh Bhoria. Application of saliva as diagnostic bio-fluid an overview. Journal of Diagnostics. 2014;1(2):13-7.

38. Fischer SG, Perez RS, Nouta J, Zuurmond WW, Scheffer PG. Oxidative Stress in Complex Regional Pain Syndrome (CRPS): No Systemically Elevated Levels of Malondialdehyde, F2-Isoprostanes and 8OHdG in a Selected Sample of Patients. Int J Mol Sci. 2013 Apr 10;14(4):7784-94 DOI: 10.3390/ijms14047784

39. Hatzipantelis ES, Karasmanis K, Perifanis V, Vlachaki E, Tziomalos K, Economou M. Combined chelation therapy with deferoxamine and deferiprone in β-thalassemia major: compliance and opinions of young thalassemic patients. Hemoglobin. 2014;38(2):111-4. DOI: 10.3109/03630269.2013.867407

40. Kochhar H, Leger CS, Leitch HA. Durable Red Blood Cell Transfusion Independence in a Patient with an MDS/MPN Overlap Syndrome Following Discontinuation of Iron Chelation Therapy. Case Reports in Hematology. 2015;2015: Article ID 253294, 7 pages.

41. Shalaby EA, Shanab SMM. Antioxidant compounds, assays of determination and mode of action. African Journal of Pharmacy and Pharmacology. 2013;7(10):528-39. DOI: 10.5897/AJPP2013.3474

42. Mohamed R, Campbell J-L, Cooper-White J, Dimeski G, Punyadeera C. The impact of saliva collection and processing methods on CRP, IgE, and Myoglobin immunoassays. Clinical and Translational Medicine. 2012;1:19. DOI: 10.1186/2001-1326-1-19

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