IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (2) 2011 Studies on the Relationship Between Chromium(III) ion and Thyroid Peroxidase Activity in Sera of Patients with Thyroid Dysfunction H. G.Hasan, T. J.Mahmood, P. A. Ismael Department of Chemistry ,College of Education, Ibin-alhaitham, University of Baghdad College of Medicine, Halwer Medical University Department of Chemistry ,College of Education Science, Salahuddin University Received in :27 October 2010 Accepted in :8 February 2011 Abstract The objective of this study was to determine the concentration of trace element chromium(III) and thyroid peroxidase activity in human serum , and to find a relationship between the concentration of chromium(III) and thyroid peroxidase activity in serum of patients with hypothyroidism, hyperthyroidism and healthy subjects. Serum thyroid peroxidase was measured by enzyme linked radioimmunoassay(ELISA) method and chromium determination was by atomic absorption spectrophotometer .Comparing the values of chromium concentration and thyroid peroxidase activity in both samples showed that there were significant positive correlations between chromium levels and thyroid peroxidase activity(P<0.01, r=0.11). The results showed that Serum thyroid peroxidase activity and chromium levels were significantly higher in hyperthyroidism patients (152.0±4.6 IU/L) (2.159±0.15 ppb) respectively than normal (51.0±1.8 IU/L) (0.378±0.024 ppb) respectively and lower than normal in hypothyroidism patients (35.0±0.31 IU/L)(0.099±0.011 ppb) respectively. Key word : Chromium(III), Thyroid peroxidase, thyroid dysfunction Introduction The biosynthesis of thyroid hormone from thyroglobulin is catalyzed by thyroid peroxidase (TPO), an integral membrane protein. TPO is also a major autoantigen in autoimmune thyroid diseases [1]. Large quantities of purified TPO are essential for elucidating its structure and understanding its role in diseases activity [2]. Failure of organification could result from deficient thyroglobulin accepter, peroxidase enzyme defect and lack of H2O2. Altaration in the process of iodide organification has been detected in almost all thyroid diseases [3]. The qualitative abnormalities in the enzyme TPO system have been proved in patients with congenital thyroid diseases, and quantitative disturbances were observed in multinodular goiter, toxic adenoma, thyroiditis, and carcinoma [4]. Trace elements are known to influence hormones at levels of action, including hormone secretion and activity and binding to target tissue [5]. Conversely, hormones influence trace element metabolism at several levels of action, including excretion and transports of trace metals [6,7,8]. Hence trace elements assay in biological fluid can be used as diagnostic or prognostic aid in patients with different hormonal disturbance alongside with other biochemical parameters[9]. Chromium(III) is an essential trace element for human. Chromium(III) is an ion needed for nutritional support for the thyroid gland to help insulin through reducing body IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (2) 2011 weight since insulin blocks phosphorylation and therefore opposes the action of epinephrine and can impede thyroid hormone production [10,11]. Chromium(III) also helps to control cholesterol levels that often elevate in hypothyroidism patients as well as control blood sugar levels which are challenged by poor adrenal and thyroid gland health [12]. Serum chromium concentration are likely to be increased above the reference range in patients with metallic joint prosthesis, longevity, increased lipid quantity, impaired glucose metabolism, hyperglycemia, and glycosuria [13,14]. The aim of this study was conducted to verify the relation of the thyroid dysfunction and TPO activity together with Cr (III) ion. Material and Method Subjects 92 patients with hyperthyroidism and 82 patients with hypothyroidism for both sexes were involved in the study they were 27-68 years of age. 92 healthy person for both sexes aged 19-54 years were participated in this study and represented the control group. Samples Collection and preparation Volume of five milliliters of venous blood from patients and healthy subjects were drawn by utilizing disposable plastic syringes and transferred into sterile test tube. The blood was allowed to clot and centrifuged at 4000 rpm for 10 minutes. Sera were separated and stored at 20 Cº until analysis . Estimation of trace element chromium Serum chromium was determined using Flame Atomic Absorption Spectrophotometer .Standards. Samples and blanks for estimation of chromium were aspirated into a ( Perkin Elmer 6000) atomic absorption spectrophotometer utilizing along-path air/acetylene burner and cathode lamp for chromium metal. Each sample was read three times at 1 second. The concentration of chromium was found by standard addition method[15,16]. Estimation of thyroid peroxidase (TPO) activity Serum thyroid peroxidase activity was measured by Enzyme Linked Immunosorbent Assay (ELISA) using ORGENTEC thyroid peroxidase (TPO) (ELISA) kit. The activity of TPO was assayed from calibration curve by interpolation . Results and Discussion The results and comparisons of serum chromium levels and TPO activity between hyperthyroidism, hypothyroidism and control groups were demonstrated in Table (1,2).There was positive correlation between serum chromium levels and TPO activity. The results of the present study revealed that serum chromium levels of hypothyroidism patients were significantly lower (0.099±0.011 ppb) than the levels in normal subjects (0.378±0.024 ppb) as shown in Table (1). A significant increase in serum chromium levels was demonstrated in hyperthyroidism patients (2.159±0.15 ppb) as compared with that of normal subjects. Chromium is an essential mineral that is required in the maintenance of our health and it is essential to the metabolism of lipids, proteins, carbohydrates and insulin regulation [17,18]. Thyroid activity may directly or indirectly affect chromium status, low thyroid function may allow increased insulin secretion resulting in chromium loss or increased insulin production suppress thyroid function[19,20]. In this study significant decrease in the levels of Cr in hypothyroidism patients were observed .One possible explanation for these finding, that gastrointestinal absorption of chromium is severely impaired in hypothyroidism subjects [21,22,23,24]. Another explanation is due to the significant influence of TSH in the variation of the concentration of chromium in normal and altered human thyroid tissue [25]. High serum chromium levels have been reported in hyperthyroidism and serum chromium concentrations have been noted to correlate well with thyroid gland activity [26]. Data obtained showed an increased serum chromium levels in patients with hyperthyroidism and positively correlation between serum chromium levels and TPO activity. Changes in body metabolic rate have been shown to be reflected in altered chromium metabolism [27]. IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (2) 2011 The increase in serum chromium levels in hyperthyroidism patients is in agreement with the studies of other researchers indicating the important role of chromium in controlling the thyroid gland function [28,29,30]. Goncharo and Ametov [31], studied the effect of chromium supplementation on the function of thyroid gland in experiment on albino male rats. The results of their study illustrated that chromium supplementation (3Mg/body weight) improve thyroid activity in albino rats . Table (2), showed that the TPO activity in hypothyroidism patients was significantly decreased (35±0.31 IU/L), probably due to a destruction that could occurs in thyroid tissue and replacement with fibroblast and lymphocyte [32]. It has been reported that serum TPO activity decreases in patients with hypothyroidism [33]. In hashimotos thyroiditis a variable degree of infiltration with lymphocytes and fibroblalsts is commonly seen in the thyroid gland, therefore, the activity of tissue enzyme is probably affected by the degree of tissue damage. The slight decrease of enzyme activity in the thyroid gland is somewhat intriguing considering the degree of tissue destruction which caused hypothyroidism [34] . Defects of TPO are both quantitative and qualitative the alter include impaired binding to heme, impaired binding to thyrogobuline or iodine substrate, abnormal localization in the erythrocyte, and abnormal susceptibility to inhibition [35]. Thyroid peroxidase gene mutations have been identified causing either abnormal thyroid peroxidase or absent enzymatic activity or complete absence of TPO protein formation [36]. As shown in table (1), TPO activity in hyperthyroidism patients was significantly elevated (152±4.6 IU/L) in comparison with the normal subjects (51±1.8 IU/L). One possible explanation for these finding , that chronic TSH stimulation leads to increased iodide binding because of increased gland peroxidase content [37], increased iodide trapping , and presumably increased H2O2 generation [38]. hence increase TPO activity [39]. Another explanation is due to the significant influence of TSH in the concentration of iodine and chromium in normal and altered thyroid tissue. Thyroid peroxidase activity was grossly elevated in toxic goiter. This may be of significance in the pathogenesis of graves diseases [40]. The relation between TPO activity and serum chromium levels is shown in Figure (2). A significant positive correlation was obtained between TPO activity and serum chromium levels in hyperthyroidism and hypothyroidism (r=0.11), when chromium levels were decreased by more than (70 %) the TPO activity was markedly decreased . Statistically significant correlation were found among indexes of chromium status and indexes of TPO. Concomitants deficiency of both key parameters are especially dangerous. In other studies, using animals chromium deficiency in rats inhibited the production of T3 and T4 [41]. Another studies revealed that chromium deficiency in animals can exert both a direct effect on the metabolic process and an indirect one disturbing iodine metabolism. The results of this study indicated that chromium deficiency in animals enhances the effect of hypothyroidism [42]. As shown in figure (3), from a total of 260 patients with thyroid diseases the most common genders are females. It can be seen from figure (2), that 92 persons were male and 170 were females. High level of chromium was found in females. Our results are consistent with other studies such as who found by Lien et al [43], that they proved thyroid disease affects female three times more than male . References 1- Dunn, J.T. and Dunn, A. D. (2001).Update on intrathyroidal iodine metabolism .Thyroid 414 -407:(5)11 2-Taur, A.M. 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Animal Feed Science and Technology, 128: 266–275 20-Sun, Y.J. ; Ramirez, J.; Woski, S.A. and Vincent, J.B. (2000): The binding of trivalent chromium to low-molecularweight chromium-binding substance (LMWCr) and the transfer of chromium from transferrin and chromium picolinate to LMWCr. Journal of Biological Inorganic Chemistry, 5: 129–136. 21-Lindemann, M.D.; Carter, S.D.;Chiba L.I., Dove, C.R.; LeMieux, F.M .and Southern, L.L. (2004) A regional evaluation of chromium tripicolinate supplementation of diets fed to reproducing sows. Journal of Animal Science, 82: 2972–2077. 22-McNamara, J.P. and Valdez, F. (2005): Adipose tissue metabolism and production responses to calcium propionate and chromium propionate. Journal of Dairy Science, 88: 498–507. 23- Bhuloka Reddy ,S.;Charles, J.M .; Kumar, R.M. ;Seetharami, S. and Anjaneyulu, C.(2002) IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (2) 2011 Trace elemental analysis of adenoma and carcinoma thyroid by PIXE method Nuclear Instruments and Method in Physics Research 196: 333-339 24-. Bhuloka Reddy, P. ; Venkateswarlu, K. ; Bhaskara Rao, V. ; Seshagiri Rao, K. Ramachandra Rao, Y. Ramakrishna, G.J and Naga Raju, Proc. AP(2001) Akad. Sci. 5 : 185. 25-Swanson, K.C.; Harmon, D.L.;Jacques, K.A.; Larson, B.T.;Richards, C.J.; Bohnert D.W. and Paton S.J. (2000) Efficacy of chromium-yeast supplementation for growing beef steers. Animal Feed and Science Technology, 86: 95–105. 26-Waylan, A.T.; O’Quinn, P.R.; Goodband, R.D.; Unruh, J.A.; Nelssen, J.L.; Woodworth J.C. and Tokach M.D. (2003) Effects of dietary additions of modified tall oil, chromium nicotinate, and L-carnitine on growth performance, carcass characteristics, and bacon characteristic of growing-finishing pigs. 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American Journal of Clinical Nutrition, 33:57–62. 32-Lee, C.R.; Yoo, C.I.; Lee, J.H. and Kang, S.K. (2002) Nasal septum perforation of welders. Industrial Health, 40: 286– 289. 31- Goncharov, A.T and Ametov, A.S(1977) thyroid gland morphological and functional indices in the experimental action of chromium .probl E ndokrinol ,23(6):59-61 33- Valenta, L.J: (1976) Thyroid peroxidase, thyroglobulin, CAMP and DNA in human thyroid. J Clin Endocrinol Metab 43:466469, 34-Masahiro, S, ; Roy, L. ; Harri, L. and Ann, M.(1984) thyroid T4–deiodinase activit in normal and abnormal human thyroid gland .Metabolism,33(4)332-336 35-Rapoport, B. and McLachlan, S. A(2001).Thyroid autoimmunity .J.Clin.Invest.108:1253- 1259. 36-Marcocci, C. and Chiovato, L.( 2000) Thyroid–directed antibodies. In: Braverman LE, Utiger RD. The Thyroid: A fundamental and clinical text. 8th ed. Philadelphia: Williams & Wilkins, p. 414-31. 37. Nagataki, S.; Uchimura, H. and Matsuyama, Y. (1973) et al: Thyrotropin and thyroidal peroxidase activity. Endocrinology 92:363-371 38-. Yamamoto, K. and DeGroot, L.J. (1974) Peroxidase and NADPHcytochrome c reductase activity during thyroid hyperplasia and involution. Endocrinology 95:606-612, 39-. Nataf, B.M.; Fragu, P. and Othman, S.B.(1978) Relationship between peroxidase activity and serum TSH. T4 and T, levels in rats in the course of iodine deficiency. Acta Endocrinol (Kbh) 88:499-505, 40-Pechova, A.; Illek, J.; Sindelar, M. and Pavlata, L. (2002): Effects of chromium supplementation on growth rate and metabolism in fattening bulls. Acta Veterinaria Brno, 71:535–541. 41-Juturu, V.; Komorowski, J.R.; Devine, J.P. and Capen, A. (2003) Absorption and excretion of chromium from orally administered chromium chloride, chromium acetatate and IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (2) 2011 chromium oxide in rats. 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Table (1): The mean ± SE of serum levels of chromium and Thyroid peroxidase(TPO) activity In hyperthyroidism patients and normal subjects P-value normal subjects hyperthyroidism patients Parameters 0.001 51.0 ±1.8 152.0± 4.6 Thyroid peroxidase (TPO) IU/L 0.003 0.35±0.0241 2.159±0.15 Chromium ppb Table(2): The mean ± SE of serum levels of chromium and Thyroid peroxidase(TPO) activity In hypothyroidism patients, and normal subjects P-value normal subjects Hypothyroidism patients Parameters 0.006 51.0 ±1.8 35.0± 0.31 Thyroid peroxidase (TPO) IU/L 0.001 0.35±0.0241 0.09±0.011 Chromium ppb Y=0.0216X – 1.2734 r =0.12 IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (2) 2011 Y=0.0028X – 0.0027 r=0.11 2011) 2( 24المجلد مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة الكروم الثالثي وفعالیة انزیم بیروكسیدیز دراسة عن العالقة ما بین تركیز ایون الدرقي في امصال المصابین باختالل الوظیفة الدرقیة ور جلیل محمود ، بروین عبد الصمد اسماعیلفحامد غفوري حسن ، طی د،ابن الهیثم -كلیة التربیة قسم الكیمیاء ، جامعة بغدا جامعة هاولیر الطبیة،كلیة الطب الدین جامعة صالح،كلیة التربیة 2010تشرین األول 27:استلم البحث في 2011شباط 8: قبل البحث في الخالصة تتلخص اهداف هذه الدراسة في تقدیر تركیز ایون العنصر الضئیل الكروم الثالثي وكذلك تقدیر فعالیة انزیم الوظیفة الدرقي، ومن ثم ایجاد عالقة تربط ما بین البیروكسیدیز الدرقي في مصول االشخاص المصابین بمرض اختالل ق مطیافیة االمتصاص الذري في تقدیر تركیز الكروم في عینات المصول المرضیة والطبیعیة كما ائت طر لمعاست. االثنین ا اوجدت النتائج ان هناك زیادة معنویة. في تقدیر فعالیة انزیم البیروكسیدیز الدرقي ELISAت تقنیة ال لمعواست وارتفاع على التوالي في ) ppb ) (152.0±4.6 IU/L 0.15±2.159(ملحوظین في مستویات الكروم وفعالیة البیروكسیدیز كما ). 0.024ppb ) (51.0± IU/L1.8±0.378(مصول المرضى المصابین بالفرط الدرقي عنه في المصل الطبیعي ة نانخفضا ا بینت النتائج ان تركیز الكروم وفعالیة البیروكسیدیز قد خفاضا ملحوظا في مصول قصور الدرقی )0.099±0.011 ppb ) (35.0±0.31 IU/L (ة . وعلى التوالي ومقارنة في امثالها عند الطبیعیین كما بینت النتائج العالق . نعند مقارنة تركیزالكروم وفعالیة البیروكسیدیز عند المرضى والطبیعیی ) P<0.001, r=0.11( المعنویة واالیجابیة