Protein thiol (µmol/L) RELATIONSHIP BETWEEN THYROID STIMULATING HORMONE AND ANTIOXIDANT STATUS IN PATIENTS WITH SUSPECTED THYROID DYSFUNCTION   A. M. S. M. Wickramarathne1,2*, R. Sivakanesan2, D. K. K. Nanayakkara3   1Post Graduate Institute of Science, University of Peradeniya 2Department of Biochemistry, Faculty of Medicine, University of Peradeniya 3 Nuclear Medicine Unit, Faculty of Medicine, University of Peradeniya Abstract Thyroid hormones are involved in the regulation of basal metabolic rate and in oxidative metabolism. In hyperthyroid conditions metabolic activities are increased and thereby there is an increase in the total consumption of oxygen. These conditions can promote formation of reactive oxygen species and other free radicals, resulting in oxidative stress and impairment of the antioxidant system. The study was conducted on antioxidant status and thyroid stimulating hormone (TSH) of patients aged between 15 and 85 years, who were referred to the Nuclear Medicine unit to assess the thyroid function from September to December 2013. At the time of sample collection patients were not on thyroxine or antithyroid drugs. The study was approved by the Ethics committee of the Postgraduate Institute of Science, University of Peradeniya. Serum TSH level was measured using immuno-radiometric assay. Antioxidant status was estimated by serum Total Antioxidant Capacity (TAC) using Ferric Reducing Ability of Plasma, and the extent of protein oxidation was assessed by the serum protein thiol concentration using Ellman’s method. The study population (n=93) had a mean TSH of 5.4±12.3 mU/L. Mean TAC of the study group was 719.1±164.5 µmol/L and mean Protein thiol was 517.7±89.5 µmol/L. There was no significant correlation between TSH and TAC (P=0.103) or TSH and protein thiol (P=0.269). In the study population, 9 (9.7%) patients were hyperthyroid, 68 (73.1%) were euthyroid while 16 (17.2%) were hypothyroid. There was a significant positive correlation between TSH and TAC (r=0.526, P=0.036) in hypothyroid patients. Majority of the study group (n=85, 91.4%) were females and 8 (8.6%) were males. Serum TAC and protein thiol concentrations of females had a significant negative correlation (r= -0.263, P=0.015). In contrast, no significant correlation was observed between TAC and protein thiol concentrations in males (r=0487, P=0.221). In the study group, 22 patients (23.7%) were aged below 35 years, 37 (39.8%) were aged between 35 to 50 years, while 34 (36.5%) were aged above 50 years. No significant correlations were observed between TSH and TAC or TSH and protein thiol in any age group. But the TAC in patients >50 years of age (466.2 µmol/L) was significantly lower than that of patients between 35 – 50 years (524.7 µmol/L). In contrast mean protein thiol significantly increased with the increase in age. The study thus concludes that, there is a significant positive correlation between TSH and TAC in hypothyroid patients. There were no significant differences in TAC and protein thiol concentrations between the hypothyroid, euthyroid and hyperthyroid patients. Introduction Thyroid hormones are involved in the regulation of basal metabolic rate and in oxidative metabolism. In hyperthyroid conditions metabolic activities are increased and thereby there is an increase in the total consumption of oxygen. These conditions can promote formation of reactive oxygen species (ROS) and other free radicals, resulting in oxidative stress and impairment of the antioxidant system (Abalovich et al., 2003). Studies have shown that thyroid hormones in excess are accompanied by increased oxidative stress and impairment of the antioxidant system (Mirela et al., 2013). Hence a study was conducted with the objective of understanding the relationship between serum thyroid stimulating hormone (TSH) and serum antioxidant levels in patients with suspected thyroid dysfunction who were currently not on treatment. Methodology Samples were collected from patients who were referred to the Nuclear Medicine unit, University of Peradeniya to assess the thyroid gland function. Patients who were not on thyroxine or antithyroid drugs at the time of sample collection were selected for the study after obtaining their informed written consent. Test subjects were both males and females and their age ranged from 15 to 85 years. A total of 93 subjects were included in the study. Ethical clearance for this study was obtained from Ethics Committee, Postgraduate Institute of Science. Serum TSH level was measured using immuno-radiometric assay method (IRMA). Oxidant stress was estimated by serum Total Antioxidant Capacity (TAC) and serum protein thiol concentration. TAC was estimated using Ferric Reducing Ability of Plasma, by reducing ferric-tripyridyltriazine (Fe3+-TPTZ) to form a blue colored ferrous- tripyridyltriazine (Fe2+-TPTZ) complex (Benzie and Strain, 1996). Protein thiol in serum was estimated using Ellman’s reagent (Koster et al., 1986). Results The study population (n=93) had a mean TSH of 5.4 ± 12.3 mU/L. Mean serum TAC was 719.1 ± 164.5 µmol/L and the mean serum protein thiol concentration was 517.7 ± 9.3 µmol/L (Table 1). There was no significant correlation between TSH and TAC (P=0.103) or TSH and protein thiol (P=0.269). Table 1. Mean, maximum and minimum values for Age, TSH, TAC and Protein thiol In the study population, 9 patients (9.7%) were hyperthyroid, 68 (73.1%) were euthyroid while 16 (17.2%) were hypothyroid. There was a significant positive correlation between TSH and TAC (r=0.526, P=0.036) in hypothyroid patients. No such correlation was observed in hyperthyroid or euthyroid patients. There was no significant difference in the mean TAC or protein concentrations between the three groups (Table 2). The protein thiol concentration was lowest in hyperthyroid patients and highest in hypothyroids even though the mean concentrations among the groups were not significantly different. Table 2. Mean values for Age, TSH, TAC and Protein thiol Majority of the study group (n=85, 91.4%) were females and 8 (8.6%) were males. Serum TAC and protein thiol concentrations of females had a significant negative correlation (r= -0.263, P=0.015). In contrast, no significant correlation was observed between TAC and protein thiol concentrations in males (r=0487, P=0.221). Discussion Mean TAC value seen in this study (719.1±164.5 µmol/L) is slightly lower than that of apparently healthy Chinese adults (1017 ± 206 µmol/L) Benzie and Strain (1996), and higher than seen in plasma samples (514.1 ± 19.1 µmol/L) of unselected Italian outpatients referred to the Institute of Clinical Physiology (Vassalle et al., 2004). Mean protein thiol concentration obtained in this study was higher than those seen in healthy controls (376 ± 64 µmol/L) in India (Suresh et al., 2010). Mean TAC in healthy Netherland males reported by Jansen and Ruskovska (2013) (1392 ± 158 µmol/L) was higher than that seen in this study while the mean protein thiol (431 ± 52 µmol/L) concentration was lower than that seen in males in the present study. This study showed a significantly negative correlation between mean TAC and protein thiol levels of females. In males there was no significant correlation. In a study done by Jansen and Ruskovska (2013), protein thiols of males have shown a negative but no significant correlation with the TAC (r = −0.008). In contrast to the present study, Rao and Prasad (2012) observed significant decrease in protein thiol levels in hyperthyroid and hypothyroid patients compared to euthyroid controls. Mean ± SD Minimum Ma ximum Age (Years) 44.7 ± 13.8 15.1 80.8 TSH(mU/L) 5.4 ± 12.3 0.1 58.0 TAC (µmol/L) 719.1 ± 164.5 360.2 1261.0 Protein thiol (µmol/L) 517.7 ± 89.5 311.6 689.3 Conclusion There was no significant difference in TAC or protein thiol concentrations between the hypothyroids, euthyroids and hyperthyroids. A significant positive correlation was observed between TSH and TAC in hypothyroid patients. References Abalovich, M., Liesuy, S., Gutierrez, S. and Repetto, M. (2003). Peripheral parameters of oxidative stress in Graves’s disease: The effect of methimazole and 131 iodine treatment. Clinical Endocrinology 59(3), 321-327. Benzie,I.F.F. & Strain, J.J. (1996).The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”:The FRAP assay. Analytical Biochemistry 239, 70-76. Jansen, E. H. J. M., and Ruskovska, T. (2013).Comparative analysis of serum (anti)oxidative status parаmeters in healthy persons. International Journal of Molecular Sciences 14, 6106-6115. Koster, J.F., Biemond, P., Swaak, A. J. G. (1986). Intracellular and extracellular sulphydryl levels in rheumatoid arthritis. Annals of the Rheumatic Diseases 45, 44-46. Mirela, P., Adriana, M. and Ileana, D. (2013). Oxidative Stress and Antioxidant Status in Hypo- and Hyperthyroidism. Available from http://dx.doi.org/10.5772/51018 (Accessed 23 February 2013). Rao, N., and Prasad, A. (2012). Erythrocyte glutathione system and plasma protein oxidation in thyroid dysfunction. Pharmacie Globale (IJCP) 1 (06). Suresh, B., Shetty, J. K. and Prakash, M. (2010). Cardiac Enzymes, Total Thiols And Lipid Peroxidation In Patients With Acute Myocardial Infarction. Journal of clinical and diagnostic research 4, 3425-3429. Vassalle, C., Masini, S., Carpeggiani, C., L'Abbate, A., Boni, C. and Zucchelli, G.C., (2004). In vivo total antioxidant capacity: comparison of two different analytical methods. Clin Chem Lab Med 42, 84-89. Age (Years) TSH (mU/L) TAC (µmol/L) Protein thiol (µmol/L) Hyperthyroid (n=9) Mean ± SD 53.3 ± 16.1 0.1222 ± 0.044 785.5 ± 151.7 496.5 ± 141.5 Range 26.90 - 71.00 0.1000 - 0.2000 640.5 - 1075.8 328.1 - 689.3 Median 55.7 0.1 780.3 483.5 Euthyroid (n=68) 44.6 ± 12.5 1.82 ± 1.060 706.6 ± 167.1 516.0 ± 79.5 16.60 - 80.80 0.400 - 4.000 360.2 - 1261.0 311.60 - 678.30 44.85 1.6 683.4 527.1 Hypothyroid (n= 16) 40.0 ± 16.3 23.38 ± 22.39 734.9 ± 159.8 536.8 ± 97.4 15.10 - 70.60 4.20 - 58.00 430.8 - 955.1 388.8 - 673.7 41.1 11.2 739.8 546.4