Role of zinc, ferritin, oxidative stress, and uric acid levels in hypothyroidism patients

Introduction

Hypothyroidism is a common condition in which the amount of thyroid hormones in the body is below normal. It affects approximately 2% of the population. Thyroid hormones are chemical messengers that help to regulate the metabolic rate. In addition to thyroid hormonal profiles. Certain additional parameters directly or indirectly linked with thyroid hormone metabolism may help to assess the thyroid status. Many of the hypothyroid subjects are found to be anemic when compared to the general population [1] . Iron plays a role in the activity of the enzyme thyroperoxidase which is involved in thyroid synthesis because the enzyme is hem-dependent, It catalyzes the oxidation of Iodide (I -) to Iodin (I +), and contributes to the iodination of tyrosin & coupling of Iodothyronine. Ferritin the storage form of Iron reflects the Iron status of the individual. In anemic subjects low levels of ferritin are found in association with impaired activity of the enzyme Thyroid peroxidase [1] . Hence ferritin may serve as an indirect indicator of thyroid status because of its role in thyroid production [2] . As thyroid hormone is associated with the regulation of the metabolism Low levels of thyroid are associated with low metabolic rate. Oxidative stress production of free radicals is directly proportionate to the metabolic rate. In hypothyroid subjects due to lower metabolic rate levels of free radicals will be low and so will the products of lipid peroxidation. Hence the levels of Malondialdehyde reflect the extent of peroxidation which is low in hypothyroid subjects.

Uric acid is said to have antioxidant activity because of its ability to remove singlet oxygen, hydroxyl, and peroxide radicals. By this property, uric acid is estimated as a parameter of antioxidant activity. Considering the above facts, among to thyroid profile, ferritin, Malondialdehyde, and uric acid estimation are included Zn + in the current study [3,4,5,21] . Zn+ is also a very important mineral in thyroid hormone metabolism and improves the anti-oxidant mechanism in humans. In hypothyroidism patients lack of Zn+ absorption leads to low levels of Zn+ is observed in hypothyroidism patients. Several minerals and trace elements are essential for normal thyroid hormone metabolism Eg: iodine, iron, selenium, zinc. Co-existing deficiencies of these elements can impair thyroid function [6,1] . Evidence from earlier studies showed a positive correlation between the serum levels of T 3 / T 4 and ferritin in individuals with thyroid abnormalities. Elevated serum ferritin levels were observed in hyperthyroid individuals, and levels decreased significantly after anti-thyroid treatment with normalization of T 3 levels. Furthermore more administration of T 3 to hypothyroid individuals produced a significant increase in the serum ferritin level [20] . Thyroid hormones stimulate erythropoiesis Severe oxidative stress produces ROS and induces uncontrolled lipid peroxidation. Lipid peroxidation is a normal phenomenon that occurs continuously at low levels in every individual. Those peroxidation reactions are toxic to cells and cell membranes. However, they are normally controlled by antioxidant mechanisms. (22) Oxygen Species (ROS) and Reactive Nitrogen Species are effectively kept in check by various levels of antioxidant defence. However when it gets exposed to adverse physiochemical, Environmental, or pathological agents such as atmospheric pollutants cigarette smoking, UV rays, radiation, toxic chemicals, and advanced glycation end products. This delicately maintained balance is shifted in favour of Pro-oxidants resulting in "Oxidative stress ". It has been implicated in the aetiology of several (>100) human diseases and in the process of aging [18,19] . Oxygen free radicals (OFR) Play a significant role in the pathogenesis of chronic renal disorders [20] . On the other hand, an improper, activation of the Oxidative process may be chronically present in pathological situations and contribute to cell and tissue injury. The mitochondrial respiratory chain represents the most powerful cellular source of oxidants in the body [7,3] . Attack of OFR on polyunsaturated fatty acids initiates lipid peroxidation which leads to alterations in biological membranes and causes progressive renal injury [4,8] . Antioxidants are substances that neutralize the free radicals or their actions. Nature has endowed each cell with an adequate protective mechanism against any harmful effects of free radicals [9] . Oxidation reactions can produce free radicals, which start a chain reaction that damages cells, then the antioxidants terminate these chain reactions by removing free radical intermediates and inhibiting other oxidant reactions by being oxidized themselves [10,11] . FRAP (Ferric Reducing Anti Oxidant Power) is used to assess the total antioxidant capacity and is considered to be a useful indicator of the system's ability to regulate damage due to ROS. Studies of total antioxidant capacity in chronic kidney disease show enhancement of lipid peroxidation and decrease of antioxidants [12] .

To prevent the damage caused by free radicals, a variety of enzymatic and non-enzymatic antioxidants are present in human serum. Due to oxidative stress, there is an increased level of pro-oxidants and depletion of antioxidants leading to tissue damage. Thyroid hormones have a strong impact on oxidative stress. They increase reactive oxygen species, while activating metabolic systems of the body. Hypothyroidism led to changes in oxidant and anti-oxidant systems [13,22] . Uric acid is a powerful antioxidant and is a scavenger of singlet oxygen and radicals. At physiological concentrations, urate reduces the oxo-heme oxidant formed by peroxide reaction with hemoglobin [14,15] . Zinc is a principal mineral for the regulation of metabolic events in humans, the thyroid hormone plays an important role in the absorption of zinc by the synthesis of metallothione in protein in the intestine. Hypothyroidism patients, have very low or lack synthesis of metallothione protein poor absorption of zinc impairs the thyroid hormone metabolism and increases the oxidative stress because of impairment of activation of antioxidant enzymes [16,17] .

Materials and Methods

This study was conducted over 6 months in the Department of Biochemistry and General Medicine at Narayana Medical College & Hospital both sexes were included and all of them were in the age group of 35 to 60 years' male and female the patients were identified and diagnosis of thyroid disorders especially in hypothyroidism in the department of endocrinology Narayana Medical College & Hospital, Nellore. We are enrolled as control from the normal individuals such as healthy both sexes. To whom so ever affected by thyroid disorders or hypothyroidism the clinical parameters of T 3 , T 4 , TSH, Zinc, Ferritin, MDA, and Uric acid as inclusion parameters and exclusion parameters are Iron and copper. Informed consent was taken for all subject's sample collection.

Sample collection

After isolation of the patients from the department of Endocrinology collection of a 5ml blood sample was allowed to clot for serum separation the serum was used for the estimation of T 3 , T 4 , TSH, Zinc, Ferritin, MDA, and Uric acid.

Estimation OF T3

Method: Enzyme-linked immune sorbent Assay (ELISA) method.

Principle [23,24,25,26] The T 3 ELISA is based on the principle of competitive binding between T 3 in a test specimen and T 3 -peroxidase conjugate for a limited number of binding sites on the anti-T 3 (sheep) coated well. Thus the amount of T 3 peroxidase conjugate bound to the well is inversely proportional to the concentration of T 3 in the specimen. After incubation of the specimen and T 3 -peroxidase conjugate unbound enzyme conjugate is removed in the equilibrium state by washing. Substrate solution is added and a blue colour develops. The intensity of colour, which changes to yellow after stopping the reaction, is inversely proportional to the amount of T 3 in the specimen.

The absorbance of the specimen is determined by using an ELISA reader, with the wavelength set at 450 nm. Specimen concentration is extrapolated from a dose-response curve generated by utilizing serum calibrators of known antigen concentrations.

Specimen

Wash procedure

Remove the adhesive strips aspirate off the contents, add wash solution, and aspirate off in 30 sec. repeat washing twice. After washing remove the remaining liquid by tapping the plate upside down on tissue paper.

Calculation

1. Plot the absorbance for each duplicate versus the corresponding T 3 concentration in pg/ml on linear graph paper. 2. To determine the concentration of T 3 of an unknown sample, locate the average absorbance of duplicates on the vertical axis of the graph, find the intersecting point on the curve, and read the concentration from the horizontal axis of the graph.

Normal values: 1.4 -4.2 pg/ml.

Estimation of T4

Principle: [27,28,29,30,31,32] The T 4 ELISA is based on the principle of competitive binding between T 4 in a test specimen and T 4 -peroxidase conjugate for a limited number of binding sites on the anti-T 4 (sheep) coated well. Thus the amount of T 4 peroxidase conjugate bound to the well is inversely proportional to the concentration of T 4 in the specimen. After incubation of the specimen and T 4 -peroxidase conjugate unbound enzyme conjugate is removed in the equilibrium state by washing. Substrate solution is added and a blue colour develops. The intensity of colour, which changes to yellow after stopping the reaction, is inversely proportional to the amount of T 4 in the specimen. The absorbance of the specimen is determined by using an ELISA reader, with the wavelength set at 450 nm. Specimen concentration is extrapolated from a dose-response curve generated by utilizing serum calibrators of known antigen concentrations.

Specimen: Serum-Do not use highly lipemic or hemolysed specimens. Principle: [33,34,35] The TSH ELISA, makes use of a highly specific monoclonal anti-TSH antibody coated on the surface of the micro wells.

Reagents

In the first incubation step, the specimen and enzyme conjugate (peroxidase-labelled anti-TSH) are mixed to form the sandwich complex which is bound to the surface of the wells by the interaction with the immobilized antibody. At the end of the incubation excess enzyme conjugate is washed out. A substrate reagent is added and the resulting colour, which turns into yellow after stopping the reaction with the stop solution is measured photometry. Absorbance increase is directly proportional to the TSH concentration in the sample. The concentration is evaluated by means of a calibration curve which is established from the calibrators supplied with the kit. Principle: [36,37,38] Anti The absorbance is read at 535 nm, using a butanol blank. Principle [39,40] Specimen 1. Serum. Estimation of Zinc [41,42] Estimation of Zinc Nitro-PAPS method (Phosphorus Adenosyl Phospho Sulfate) Zinc in an alkaline medium reacts with nitro-PAPS to form a purple-coloured complex.

Specimen

Reagents

The intensity of the complex formed is directly proportional to the amount of zinc present in the sample. Reference values Zinc = 66 -106 mcg/dL

Statistical Analysis

The data was analyzed using the statistical software SPSS12. Version. Values are expressed as mean ± SD comparison of continuous variables was done. Using PEM subjects 't' test correlation and in case of Skeweeds Ranle correlation test was used p-value and t value.

Results

Table 1: Show the mean standard deviation and P value of all the investigations of the study done in both Hypothyroid subjects and control subjects. The two-tailed p-value is less than 0.001 by conventional criteria, this difference is considered to be extremely statistically significant.

S. No

Confidence interval

The mean of hypothyroidism subjects minus controls equals intermediate values used in calculations T 3 Z= -2.771, T 4 Z = -7.717, TSH Z = 12.603

Ferritin p value statistically significant

The two-tailed p-value is less than 0.001 by conventional criteria, this difference is considered to be extremely statistically significant.

Confidence interval

The mean of hypothyroidism subjects minus controls equals intermediate values used in calculations Ferritin Z = -1.135

MDA p value statistically significant

The two-tailed p-value is less than 0.001 by conventional criteria, this difference is considered to be extremely statistically significant.

Confidence interval

The mean of hypothyroidism subjects minus controls equals intermediate values used in calculations MDA Z = -7.718

Uric Acid p value statistically significant

The two-tailed p-value is less than 0.001 by conventional criteria, this difference is considered to be extremely statistically significant.

Confidence interval

The mean of hypothyroidism subjects minus controls equals intermediate values used in calculations Uric Acid Z = 4.513

Zinc p value statistically significant

The two-tailed p-value is less than 0.001 by conventional criteria, this difference is considered to be extremely statistically significant.

Confidence interval

The mean of hypothyroidism subjects minus controls equals intermediate values used in calculations Zinc Z = 2.210

Discussion

Ferritin and Hypothyroidism

Hypothyroidism is a common disease state affecting adolescent and adult people, women are affected more relatively. It is not attributable to a single course. Metabolic, nutritional immunological, and genetic etiological factors were proposed. Many groups of scientists and physicians worked on this problem and proposed their observations and concepts based on experimental, Clinical, and epidemiological studies. One of the observations is thyroid hormone status is associated with the ferritin status of serum and tissues and iron-deficient anemia may be a cause of hypothyroidism. This proposal stemmed from the fact that the thyroid peroxidase enzyme which oxidizes iodide to a higher valence state before it is incorporated to form thyroid hormones is a heme-containing enzyme and hence iron plays a role in thyroid hormone synthesis.

In view of the foregoing many scientific groups worked on it. Notable observations are as follows.

Shakir et al. (2007) [7] observed co-existence of iron deficiency anemia with hypothyroidism. Sanjay et al. (2002) [19] reported that iron deficiency impaired thyroid metabolism. Peter J. Leedman (2007) [11] et al. showed a positive correlation between thyroid hormone and ferritin in serum. Effterkhan MH et al. (2006) [14] observed iron deficiency affected thyroid status in adolescent girls. Mahammed Hasan et al. (2008) [9] showed reduced thyroid peroxidase activity in thyroid-deficient rats. Liver ferritin synthesis rate was reduced by 36% in hypothyroid rats (Deshpande et al.,1993) [17] . Zimmerman M et al. (2000) [13] showed an improved thyroid profile in iron-deficient adolescent girls after iron supplementation. In view of the above available data, the present study was undertaken to evaluate the association of ferritin status with hypothyroidism. The studies were conducted in 30 hypothyroid patients. 20 age and sex matched controls were taken for study. The mean value of serum ferritin hypothyroid patients is 63.73 ng/ml against 92.54 ng/ml in control subjects. It amounts to about 30% lower value. The P value was not statistically significant as the sample size was small. Low ferritin values were observed in about 70% of cases (17 out of 30 cases). This observation indicates the correlation of iron metabolism with thyroid hormone synthesis. Considering the observations given by many groups it has to be searched whether it is due to nutritional deficiency of iron, or due to a defect in the synthesis of ferritin by the liver any genetic expression of ferritin synthesis is involved. A broader study with a greater number of hypothyroid subjects is required to confirm the association of iron with thyroid hormones.

Thyroid and Oxidative stress

Hypothyroidism brings down the metabolic rate and there will be low levels of electron transport chain activity. Iron deficiency may also contribute to it as the cytochromes, the main components of ETC are iron-containing heme compounds. Hence there is a likelihood of less release of superoxide and low lipid peroxidation. The observation was confirmed by many scientific groups. Rasin Magukot et al. (2000) [24] demonstrated higher lipid peroxidation in hyperthyroid rats. Decreased free radical and lipid peroxidation was shown by Vendetti .P. et al. (1997) [25] and Veronica M.et al. (2005) [26] . Joe Chehad et al. (1999) [30] observed low MDA and MDA Proteins in hypothyroid rats. Chowdary. S. et al. [31] suggested based on a study that alteration in thyroid hormone levels affects the antioxidant defence system. In the present study, the mean value of MDA is 4.11 m mol/L in hypothyroid patients as against 5.17 m mol/L in control subjects. Though the values are not statistically significant, it necessitates a further study with broader sampling.

Hypothyroidism and uric acid

Uric acid is an end product of purine metabolism. In hypothyroidism, because of low metabolic profile ADP levels will be more as against ATP. Hence Adenine is oxidized through the Xanthine oxidase system and liberates more uric acid. Uric acid is a powerful antioxidant and is a scavenger of singlet oxygen and radicals. Chowdary G B et al. (1999) [31] , Bruce N. Aimes et al. (1981) [32] , and Yokogoshi et al. (1996) [33] observed elevated serum uric acid levels in hypothyroid subjects.

In the present study mean serum uric acid level is 5.45 mg/dl against 4.04 mg/ dl in controls which is statistically significant. Hence studying serum uric acid levels in hypothyroid patients may be used as an associated biochemical parameter to follow the course of the disease. It may be also used to assess whether any myopathic changes are going on, as seen in advanced hypothyroidism. Zn+ is also a very important mineral in thyroid hormone metabolism and improves the anti-oxidant mechanism in humans. In hypothyroidism patients lack of Zn+ absorption leads to low levels of Zn+ is observed in hypothyroidism patients Conclusion 1. Ferritin levels are low in hypothyroid patients suggesting the role of nutritional iron for thyroid hormone formation. 2. Malondialdehyde, an indicator of lipid peroxidation is also low in hypothyroidism which can be considered as one of the associated parameters to be considered for diagnosis and prognosis. 3. Uric acid levels within physiological limits are elevated in hypothyroid patients. It may help to observe the setin of myopathic changes. 4. An extended study with broader sampling is required to strongly support the observations. 5. The dietary zinc is not observed from the intestine due to a lack of thyroid hormone-associated synthesis of metallothionein protein, the protein synthesis is also enhanced by GIT motility. 6. Due to hypothyroidism condition low GIT motility causes the impair synthesis of zinc absorption carrier protein. This results in acrodermatitis enteropathica is seen in hypothyroid patients. This leads to loss of appetite and impairment of antioxidant mechanism which results in MDA levels are elevated.

References

1. Zimmermann MB, Burgi H, Hurrell RF. Iron deficiency predicts poor maternal thyroid status during pregnancy. Journal of Clinical Endocrinology & Metabolism. 2007 Sep;92(9):3446-3450.
2. Zimmermann MB, Kohrle J. The impact of iron and selenium deficiencies on iodine and thyroid metabolism: Biochemistry and relevance to public health. Thyroid. 2002 Oct;12(10):867-878.
3. Vydyborets' SV. Iron deficiency anemia as a hematological mask of myxedema. Likars'ka Sprava. 1999 Jun;(4):148-150.
4. Rosenzweig PH, Volpe SL. Iron thermoregulation and metabolic rate. Chung Hua Yu Fang I Hsueh Tsa Chih. 1996 Nov;30(6):351-353.
5. Zimmermann MB, Chaouki N, Hurrell RF. Iron deficiency due to consumption of a habitual diet low in bioavailable iron: a longitudinal cohort study in Moroccan children. Journal of Nutrition. 2005 Dec;135(12):3026-3032.
6. Shakir KM, Turton D, Aprill BS, Drake AS, Elisold JF. Anemia: a cause of intolerance to thyroxine sodium. Mayo Clinic Proceedings. 2000 Feb;75(2):189-192.
7. Dabbaghmanesh MH, Abdols, Sadegholvaad M, Hadi F, Ranjbaromrani GH. Iron deficiency reduces thyroid peroxidase activity in rats. Iranian Journal of Medical Sciences. 2008 Nov;33(4):157-161.
8. Hess SY, Zimmermann MB, Arnold M, Langhans W, Hurrell RF. The role of iron deficiency in persistent goiter. Journal of Nutrition. 2002 Aug;132(8):19511955.
9. Leedman PJ, Stein AR, Chin WW, Rogers JT. Thyroid hormone modulates the interaction between iron regulatory proteins and the ferritin mRNA ironresponsive element. Experimental and Clinical Endocrinology & Diabetes. 2007 Jun;115(6):522-526.
10. Mathus N, Joshi SC, Mathus S. Effect of dietary iron deficiency anemia on TSH and peripartum thyroid function. Endocrine Abstracts. 2006;12:123.
11. Zimmermann MB, Adoup M, Torresani T, Zeder C, Hurrell RF. Iron supplementation in goitrous, irondeficient children improves their response to oral iodized oil. American Journal of Clinical Nutrition. 2000 Jan;71(1):88-93.
12. Efterkhani MH, Keshavarz SA, Jalali M, Elguero E, Eshraghian MR, Simondon KB. The relationship between iron status and thyroid hormone concentration in iron-deficient adolescent Iranian girls. Asia Pacific Journal of Clinical Nutrition. 2006;15(1):50-55.
13. Azizi F, Mirhadi M, Sheikholeslam R, Tohidi M, Rastmanesh R. The relationship between serum ferritin and goiter, urinary iodine, and thyroid hormone concentration. International Journal for Vitamin and Nutrition Research. 2002;72(5):296-299.
14. Tienboon P, Unachak K. Iron deficiency anemia in childhood and thyroid function. Asia Pacific Journal of Clinical Nutrition. 2003;12(2):198-202.
15. Desahpanda UR, Nadkarni GD. Relation between thyroid status and ferritin metabolism in rats. Clinical Investigations. 1993 Dec;72(1):26-29.
16. Fein HG, Rivlin RS. Anemia in thyroid diseases. Medical Clinics of North America. 1975 Sep;59(5):1133-1145.
17. Das KC, Mukherjee M, Shankar TK, Dash RJ, Rastogi GK. Erythropoiesis and erythropoietin in hypo-and hyperthyroidism. Journal of Clinical Endocrinology & Metabolism. 1975;40(2):211-220.
18. Beard JL, Brigham DE, Kelley SK, Green MH. Plasma thyroid hormone kinetics are altered in iron-deficient rats. Journal of Nutrition. 1998 Aug;128(8):1401-1408.
19. Sathyanarayana U. Minerals. 5 th ed.; 2016. p. 244.
20. Mayulkoc R, Kasim A, Bultci A, Ozturk E, Aydin L, Junler I. Hyperthyroidism causes lipid peroxidation in kidney and testis tissues of rats; protective role of melatonin. Neuroendocrinology Letters. 2005;26(6):806-810.
21. Pedersen K. Serum lipids and proteins in pregnancy. Scandinavian Journal of Clinical and Laboratory Investigation. 1974;34:24-30.
22. John R, Shankland D. Clinical significance of abnormal laboratory values. Clinical Chemistry. 1990;36:470475.
23. Wenzel KW. Metabolic implications of thyroid hormone levels. Metabolism. 1981;30:717-722.
24. Wosilait WD. Serum levels of thyroid hormones in various diseases. Research Communications in Chemical Pathology and Pharmacology. 1977;16:54150.
25. Wenzel KW. Metabolic effects of thyroid hormones. Metabolism. 1981;30:717-722.
26. Konishi J, Kishi H, Kajiwara K, et al.. Clinical significance of thyroid hormone levels in various conditions. Clinical Chemistry. 1982;28:1389-1394.
27. Lundberg PR, Ekman R, Hällström T, et al.. The role of thyroid hormones in metabolic regulation. Clinical Chemistry. 1982;28:1241-1245.
28. Bhagat C, Kaul B, Ghosh S, et al.. Serum thyroid hormone levels and their clinical implications. Clinical Chemistry. 1983;29:1324-13230.
29. Norden AGW, Zetterström R, Källner A, et al.. Accuracy and clinical interpretation of thyroid function tests. Clinical Chemistry. 1997;43:957-963.
30. Banker SB. Determination of protein-bound iodine. Journal of Biological Chemistry. 1948;173:175-80.
31. Chopra IJ, Rebar RW, Solomon DH, et al.. A radioimmunoassay of thyrotropin (TSH). Journal of Clinical Endocrinology & Metabolism. 1971;33:865873.
32. Young DS, Culler FD, Becker DM. Effects of drugs on clinical laboratory tests. Clinical Chemistry. 1975;21:3660-3667.
33. Beard JL. Iron biology in immune function, muscle metabolism, and neuronal functioning. Journal of Nutrition. 2001;131(2):5685-5705.
34. Dawson DW, O'Brien KM, Birkenmeier EH, et al.. The accuracy and clinical interpretation of serum ferritin assays. Clinical Laboratory Haematology. 1992;14(1):47-52.
35. Powell LW, Adams PC, Barton JC, et al.. Diagnosis of hemochromatosis. Annals of Internal Medicine. 1998;129:925-931.
36. Barham D, Trinder P. An improved method for the determination of blood glucose. Analyst. 1972;97:1425.
37. Fossati P, Principe L, Berti G. Serum triglycerides determination using a colorimetric enzymatic method. Clinical Chemistry. 1980;26(2):227-229.
38. Veldee MS. Nutritional assessment, therapy, and monitoring. In: Tietz NW, editor. Tietz Textbook of Clinical Chemistry. 3 rd ed. Philadelphia: WB Saunders; 1998. p. 1359-1394.