|Year : 2018 | Volume
| Issue : 2 | Page : 70-74
Persistence of oxidative stress in newly diagnosed hypothyroid patients despite effective thyroxin therapy
Nivedita Nanda1, Zachariah Bobby1, Abdoul Hamide2
1 Department of Biochemistry, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India
2 Department of Medicine, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India
|Date of Submission||02-May-2018|
|Date of Decision||28-May-2018|
|Date of Acceptance||31-May-2018|
|Date of Web Publication||11-Sep-2018|
Dr. Zachariah Bobby
Department of Biochemistry, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry - 605 006
Source of Support: None, Conflict of Interest: None
Background and Aim: We have earlier reported increased oxidative stress (OS) in newly diagnosed hypothyroid patients. However, the comprehensive effect of thyroxin supplementation on OS, insulin resistance, inflammation, and glycation levels has not been analyzed. Therefore, in the present study, we have analyzed the effect of thyroxin therapy in newly diagnosed hypothyroid patients on OS and various biochemical markers after normalization of thyroid profile. Methods: Out of 67 recently diagnosed primary hypothyroid patients, 37 patients were recruited for this study based on the criteria of strict adherence to thyroxin treatment protocol and regular follow-up. Venous blood samples were analyzed before and 6 months after initiation of therapy for glucose, thyroid and lipid profiles, insulin, ultrasensitive C-reactive protein (usCRP), and anti-thyroperoxidase (TPO) antibody. Antioxidants such as glutathione, glutathione peroxidase, catalase, and glutathione S-transferase, and oxidized products such as malondialdehyde (MDA), and protein carbonyl (PCO) levels were analyzed as parameters of OS. HbA1 and fructosamine were assayed as glycation indices, and lipid risk factor for coronary artery disease was calculated from lipid profile. The parameters were re-assayed 6 months later after normalization of thyroid profile. Results: OS (MDA; P < 0.01 and PCO; P < 0.01) did not come back to normal level despite attainment of normal thyroid profile following treatment. Dyslipidemia (P < 0.05) and inflammation (P < 0.05) were significantly associated with OS. Furthermore, levels of triglyceride, anti-TPO antibody, and usCRP were higher in patients even after successful treatment. Conclusion: OS in treated hypothyroid patients despite normalization of thyroid profile persists longer which could partly be due to the residual inflammation and/or dyslipidemia.
Keywords: Dyslipidemia, hypothyroidism, inflammation, oxidative stress, treatment
|How to cite this article:|
Nanda N, Bobby Z, Hamide A. Persistence of oxidative stress in newly diagnosed hypothyroid patients despite effective thyroxin therapy. Int J Clin Exp Physiol 2018;5:70-4
|How to cite this URL:|
Nanda N, Bobby Z, Hamide A. Persistence of oxidative stress in newly diagnosed hypothyroid patients despite effective thyroxin therapy. Int J Clin Exp Physiol [serial online] 2018 [cited 2018 Nov 14];5:70-4. Available from: http://www.ijcep.org/text.asp?2018/5/2/70/241035
| Introduction|| |
Hypothyroidism in India is among the most common endocrine disorders. Various nationwide studies reveal that thyroid disorder is on the rise, particularly in adolescents and young adults of India. On the long run, hypothyroidism associated with chronic complications such as hyperlipidemia, secondary obesity, and atherosclerotic cardiovascular disease. The major component of the body is protein and lipids. Therefore, oxidative damage to body protein and lipids due to ensuing oxidative stress (OS) can exert its long-term health effects. We earlier reported the presence of OS in our studies  which corroborates with other studies., Hypothyroidism is a treatable endocrine condition where thyroid profile normalizes with proper treatment, and patients need to review their thyroid profile at regular intervals, thereafter. However, there are reports available which suggest that thyroid-stimulating hormone (TSH) level and hyperlipidemia in hypothyroidism normalize slowly with thyroxin replacement therapy., OS is known to be associated with obesity, hyperlipidemia, atherosclerosis, etc. Furthermore, one of the previous reports suggests that treatment with thyroxin itself produces OS.
Through this study, we intended to analyze whether thyroxin therapy normalizes OS parameters along with thyroid profile, and if not, then what are the plausible factors responsible for the persistence of OS.
| Materials and Methods|| |
Participants and study design
Clinically diagnosed primary hypothyroid patients were contacted from the Outpatient Services of Department of Medicine, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), for this study. A total of 37 patients, of which 26 were females and 11 males, were recruited for the study. All the patients had a TSH level >10 μU/ml. The newly diagnosed hypothyroid patients who were not on any medication were included in this study before their thyroxin supplementation. Hypothyroid patients already on treatment for the disease, patients taking lipid-lowering drugs or antioxidant vitamin supplements, smokers, alcoholics, hypertensives, diabetics, patients with impaired fasting glucose, pregnant women, women on hormone replacement therapy, and patients suffering from diseases other than hypothyroidism, also patients with a family history of diabetes mellitus were excluded from the study. Two female patients had a family history of thyroid dysfunction, and two other had attended menopause. Thirty-six healthy, euthyroid participants of similar age and gender were taken as controls. Control group consisted of clinically screened euthyroid participants without any infectious or inflammatory disease or chronic ailments. The study was approved by the Research Council and Human Ethics Committee of JIPMER. Written informed consent was obtained from all the participants of the study groups before their enrollment into this study.
Venous blood from the antecubital vein was collected from patients after overnight fast (before they started L-thyroxin therapy). Three millimeters of blood was collected in ethylenediaminetetraacetic acid (EDTA) bottle and the rest was kept aside for serum separation. The whole blood was used for the immediate assay of reduced glutathione (GSH) and hemoglobin (Hb). The red blood cells were separated by washing the EDTA whole blood thrice (5000 rpm for 5 min each) at 4°C using a cooling centrifuge (Kubota 6930), with cold normal saline. The packed cells were used for the subsequent analysis of erythrocyte antioxidant enzymes. Serum samples were separated from the blood collected in a separate tube and part of it was used for the immediate estimation of glucose and lipid profile. Remaining serum and plasma samples were stored in different vials for the estimation of various parameters by refrigeration at −40°C till the estimations of thyroid profile, protein carbonyls, malondialdehyde (MDA), ultrasensitive C-reactive protein (usCRP), and anti-thyroperoxidase (TPO) antibody were carried out.
Thyroid profile was assessed by estimation of serum T3, T4, and TSH. T3 and T4 were assayed by radioimmunoassay and TSH by immunoradiometric kits procured from Bhaba Atomic Research Center, Mumbai, India. The intra- and inter-assay coefficient of variations was 3.3% and 7.3%, respectively, for T3; 4.7% and 8.2%, respectively, for T4; and 3.6% and 7.8%, respectively, for TSH assay.
Glucose, insulin, and lipid profile
Fasting serum glucose was estimated by glucose oxidase method using kits from Dr. Reddy's Lab (Hyderabad, India). Insulin was estimated using IRMA kit for human following manufacture's (Immunotech, Beckman Coulter Co, Czech Republic) instructions. Lipid profile was estimated by enzymatic method using kits from Biocon (Germany) for total cholesterol and Accurex (Mumbai, India) for triglyceride (TG). High-density lipoprotein (HDL) cholesterol-precipitating reagent and enzymes were obtained from Agappe diagnostics (Thane, India). Low-density lipoprotein (LDL) cholesterol was calculated employing the Fridewald formula, as the total TG level was below 400 mg/dl for the cases. All these analyses were performed using a calibrated autoanalyzer (550 Express Plus, Ciba Corning Diagnostics, Oberlin, OH, USA).
Calculation of lipid risk factors
Various lipid risk factors of atherosclerosis such as non-HDL cholesterol, total cholesterol/HDL-cholesterol, triacylglycerol/HDL cholesterol, LDL-cholesterol/HDL-cholesterol, and atherogenic index (AI) were calculated from the estimated lipid profile.,, The AI was expressed as log10(TG/HDL-C) where TG and HDL-C were expressed in mM/L.
Assay of erythrocyte antioxidants
Whole blood reduced GSH, glutathione peroxidase (GPx), glutathione S-transferase, and catalase activities were assayed using the standard protocol described in detail previously.
Assay of serum markers of oxidation
Protein carbonyl (PCO) was estimated as a marker of protein oxidation by the 2,4-dinitrophenylhydrazine by Levine. It was expressed as nmol/mg protein. Thiobarbituric acid reactive fraction of MDA was measured as an estimate of lipid peroxidation expressed as mM/L of serum.
Assay of markers of glycation, autoimmunity, and inflammation
Glycated Hb expressed as HbA1 and glycated albumin expressed as fructosamine were assayed by ion exchange chromatography columns (Biocon, Vohl-Marienhagen, Germany) and by p-iodonitrotetrazolium violet kinetic method using Riachem kit (Hemagen Diagnostics, San Diego, CA, USA), respectively. Anti-TPO antibody was assayed by ELISA (Varelisa, Pharmacia and Upjohn, Germany) and usCRP by turbidimetric immunoassay kit adapted to autoanalyzer (Aptec Diagnostics, Belgium), respectively.
Statistical analysis of data
All parameters are expressed as a mean ± standard deviation. Statistical analyses were performed using the IBM SPSS version 13 (Chicago Inc, IL, USA) program. Normality of the data distribution was checked by Kolmogorov–Smirnov test. Significance of the differences between control and test groups before and after therapy was done by one-way ANOVA followed by Tukey-Kramer post hoc test. The association between MDA and various parameters was done by Pearson's correlation analysis. The P < 0.05 was considered statistically significant.
| Results|| |
The changes in anthropological parameters, lipid profile, OS parameters, and its complications in patients treated for 6 months with thyroxin are included in [Table 1]. OS parameters, parameters of inflammation, autoimmunity, glycation, and insulin resistance are enlisted in [Table 2].
|Table 1: Comparison of anthropological parameters and routine biochemical parameters of hypothyroid patients before and after (thyroxin therapy for 6 months) treatment with euthyroid control subjects|
Click here to view
|Table 2: Comparison of oxidative stress parameters, markers of insulin resistance, inflammation, autoimmunity, and glycation indices of hypothyroid patients before and after (thyroxin therapy for 6 months) treatment with euthyroid control subjects|
Click here to view
With regular treatment, euthyroidism was attained in the study group in about 6 months. It was also accompanied by complete reversal of previously increased levels of body mass index (BMI), thyroid profile, lipid profile (except TG), and non-HDL cholesterol [Table 1]. Level of all these parameters in the study group after treatment was not different from control group. The thyroxin treatment for 6 months reversed the levels of coronary lipid risk factors, Non-HDL-C, and LDL-C/HDL-C levels to that of the healthy control. However, despite significant decrease, other lipid risk factors such as TG/HDL-C, TC/HDL-C, AI were still higher than the mean of the control group.
Similarly, the treatment resulted in complete normalization in the levels of antioxidant enzymes such as GSH, GPx, and catalase similar to the level of the healthy controls [Table 2]. On the other hand, there was no statistical difference in insulin resistance (Homeostasis Model Assessment-Insulin Resistance [HOMA-IR]), protein glycation (HbA1c and fructosamine), and autoimmunity (Anti-TPO) among the three groups.
Despite a significant reduction in OS parameters (MDA and PCO) and marker of inflammation (usCRP), these levels did not reach the healthy control levels [Table 2]. Especially usCRP level despite significant decrease still remained higher than control [Table 2]. Furthermore, there was a significant positive correlation between CRP and MDA even after treatment [Table 3].
|Table 3: Correlation of malondialdehyde with thyroid-stimulating hormone, protein carbonylation, lipid profile, lipid risk factors, anti-thyroperoxidase Ab, and ultrasensitive C-reactive protein in hypothyroid patients after attainment of euthyroid state following thyroxin therapy for 6 months (n=37)|
Click here to view
| Discussion|| |
As a result of the treatment with thyroxin, the thyroid profile was reverted to that of the healthy control group. Although the BMI, TC, and LDL-C levels of the patients also reverted to the level of the healthy control, the posttreatment values for the other lipid risk factors such as TG/HDL-C, TC/HDL-C, and AI were still higher than the healthy controls. These results corroborate with previous reports.,
This could be due to the continuation of some inflammatory components and some degrees of hyperlipidemia. Our findings corroborate with the report of a recently conducted study in which MDA was found to be decreased but still remained high compared to control following 6 months of treatment., However, previous studies did not study the battery of antioxidant enzymes unlike us. For the first time in our study, four major antioxidants and protein carbonyl (PCO), a sensitive marker of protein oxidation, were analyzed before and after treatment.
The increased serum protein carbonyl level in hypothyroid patients had a positive correlation with the increased MDA level [Table 3]. When products of lipid peroxidation such as 4-hydroxy nonenal and MDA covalently attach to the amino acid residues of proteins, they lead to oxidative modification. Therefore, we postulate that low-grade OS associated with hypothyroidism if persists for a prolonged period, the resulting oxidative modifications to body lipids and proteins might be deleterious in these participants.
Baskol et al. analyzed only paraoxonase which is associated with HDL apolipoprotein. Furthermore, they did not analyze for the effect of treatment on inflammatory marker, glycated proteins, and autoimmune parameter and insulin resistance in their group of patients. In our study, the response of glycated proteins, anti-TPO antibody titer, and insulin resistance (HOMA-IR) was analogous to the euthyroid effect of thyroxin treatment. However, posttreatment decrease in usCRP, measured as an inflammatory marker, continued to be higher when compared to the control value [Table 2]. The regular marker of inflammation called usCRP, also known as hsCRP, is also recognized as an independent marker of cardiovascular disease. Moreover, CRP as an inflammatory mediator is recently recognized as an adjunctive marker for the global assessment of cardiovascular risk.,, Therefore, a higher level of usCRP in our study group despite regular treatment and normalization of thyroid profile indicates the persistence of the risk for coronary artery disease.
One of the previous reports suggests that treatment with thyroxin itself produces OS. Flynn et al. in 2006 reported that, despite treatment in primary hypothyroidism, patients are still at increased risk of morbidity associated with various circulatory disease, ischemic heart disease, dysrhythmias, cerebrovascular diseases, etc. However, the present study is the first of its kind to assess the level of OS in hypothyroidism estimating parameters of oxidant and antioxidant parameters before and after normalization of thyroid profile along with markers of inflammation, lipid risk factors, and other indices such as insulin resistance and autoimmunity. Furthermore, the analysis of correlations with parameters such as fructosamine, PCO, CRP, TPO, and us CRP before and after treatment has not been done in any prior study. Therefore, our study attempts at a comprehensive analysis of various factors associated with OS which was found to be persisting in hypothyroid patients who had attained thyroid profile due to their sincere compliance with treatment protocol. These results suggest that, despite normalization of TSH following prescribed thyroxin treatment, OS in hypothyroidism persists for a longer period. From the results, we also postulate that OS in these posttreatment euthyroid participants may considerably be contributed by dyslipidemia and/or inflammation.
| Conclusion|| |
Hypothyroidism is a chronic disease and requires prolonged therapy for full recovery. In view of negative implications of OS, dyslipidemia, and inflammation on health, we suggest that regular monitoring of lipid profile, inflammatory markers, and OS status should be considered in hypothyroidism management. In addition, there is a need to complement the traditional treatment of these patients with antioxidants and lifestyle modification to bring down the ill effects of persisting OS.
Informed consent was obtained from each participant after they voluntarily agreed to participate in this study.
We acknowledge the research grant from Indian Council of Medical Research in the form of junior research fellowship followed by senior research fellowship to Dr. Nivedita Nanda and as adhoc research grant to Dr. Zachariah Bobby. We also acknowledge the technical assistants from Mr. R. Vengattraman for RIA and ELISA assays.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Kochupillai N. Clinical endocrinology in India. Curr Sci 2000;79:1061-7.
Nanda N, Bobby Z, Hamide A, Koner BC, Sridhar MG. Association between oxidative stress and coronary lipid risk factors in hypothyroid women is independent of body mass index. Metabolism 2007;56:1350-5.
Sarandöl E, Taş S, Dirican M, Serdar Z. Oxidative stress and serum paraoxonase activity in experimental hypothyroidism: Effect of Vitamin E supplementation. Cell Biochem Funct 2005;23:1-8.
Marjani A, Mansourian AR, Ghaemi EO, Ahmadi A, Khori V. Lipid peroxidation in the serum of hypothyroid patients (In Gorgan – South East of Caspian Sea). Asian J Cell Biol 2008;3:47-50.
Jameson JL, Weetman AP. Disorders of the thyroid gland. In: Kasper DL, Braunwald E, Fauci AS, Hauser SL, Longo DL, Jameson JL, et al
. editors. Harrison's Principles of Internal Medicine. 16th
ed. Vol. 2. New York: McGraw-Hill; 2005. p. 2112.
Mishkel MA, Crowther SM. Hypothyroidism, an important cause of reversible hyperlipidemia. Clin Chim Acta 1977;74:139-51.
Yesilbursa D, Serdar Z, Serdar A, Sarac M, Coskun S, Jale C, et al.
Lipid peroxides in obese patients and effects of weight loss with orlistat on lipid peroxides levels. Int J Obes (Lond) 2005;29:142-5.
Sutken E, Inal M, Ozdemir F. Effects of vitamin E and gemfibrozil on lipid profiles, lipid peroxidation and antioxidant status in the elderly and young hyperlipidemic subjects. Saudi Med J 2006;27:453-9.
Heitzer T, Schlinzig T, Krohn K, Meinertz T, Münzel T. Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease. Circulation 2001;104:2673-8.
Gredilla R, López Torres M, Portero-Otín M, Pamplona R, Barja G. Influence of hyper – And hypothyroidism on lipid peroxidation, unsaturation of phospholipids, glutathione system and oxidative damage to nuclear and mitochondrial DNA in mice skeletal muscle. Mol Cell Biochem 2001;221:41-8.
Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972;18:499-502.
Grover SA, Coupal L, Hu XP. Identifying adults at increased risk of coronary disease. How well do the current cholesterol guidelines work? JAMA 1995;274:801-6.
Wallach J: Interpretation of Laboratory Tests. 6th
ed. Philadelphia: Lippincott-Raven: Little Brown and company; 1996. p. 482.
Ridker PM. Evaluating novel cardiovascular risk factors: Can we better predict heart attacks? Ann Intern Med 1999;130:933-7.
Levine RL, Williams JA, Stadtman ER, Shacter E. Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol 1994;233:346-57.
Satoh K. Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method. Clin Chim Acta 1978;90:37-43.
Erdamar H, Demirci H, Yaman H, Erbil MK, Yakar T, Sancak B, et al.
The effect of hypothyroidism, hyperthyroidism, and their treatment on parameters of oxidative stress and antioxidant status. Clin Chem Lab Med 2008;46:1004-10.
Baskol G, Atmaca H, Tanriverdi F, Baskol M, Kocer D, Bayram F, et al.
Oxidative stress and enzymatic antioxidant status in patients with hypothyroidism before and after treatment. Exp Clin Endocrinol Diabetes 2007;115:522-6.
Dalle-Donne I, Rossi R, Giustarini D, Milzani A, Colombo R. Protein carbonyl groups as biomarkers of oxidative stress. Clin Chim Acta 2003;329:23-38.
de Ferranti SD, Rifai N. C-reactive protein: A nontraditional serum marker of cardiovascular risk. Cardiovasc Pathol 2007;16:14-21.
Ridker PM, Rifai N, Cook NR, Bradwin G, Buring JE. Non-HDL cholesterol, apolipoproteins A-I and B100, standard lipid measures, lipid ratios, and CRP as risk factors for cardiovascular disease in women. JAMA 2005;294:326-33.
Lagrand WK, Visser CA, Hermens WT, Niessen HW, Verheugt FW, Wolbink GJ, et al.
C-reactive protein as a cardiovascular risk factor: More than an epiphenomenon? Circulation 1999;100:96-102.
Flynn RW, Macdonald TM, Jung RT, Morris AD, Leese GP. Mortality and vascular outcomes in patients treated for thyroid dysfunction. J Clin Endocrinol Metab 2006;91:2159-64.
[Table 1], [Table 2], [Table 3]