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Abstract

Objectives:

The aim of this study was to explore the oxidative stress profile in hashitoxicosis (HTX) and to compare it with that of healthy subjects.

Patients and methods:

Spectrophotometric methods were used to evaluate the oxidative stress markers. The selenium level was investigated by atomic absorption.

Results:

High levels of thiobarbituric acid reactive species (TBARS) and conjugated dienes were found in HTX patients (p = 0.034 and p = 0.043, respectively) compared with healthy controls. For antioxidant enzymes, superoxide dismutase (SOD) and catalase activities increased, whereas that of glutathione peroxidase (GPx) decreased (p = 0.000, p = 0.014, p = 0.000, respectively) compared with controls. A reduction in the level of selenium (p = 0.029) and thiol groups (p = 0.008) were shown in patients; however, levels of carbonyl group and malondialdehyde (MDA) protein adducts decreased (p = 0.000) compared with controls. Positive correlation was shown between levels of free thyroxine (FT4) and TBARS (r = 0.711, p = 0.048) and between FT4 level and SOD activity (r = 0.713, p = 0.047). Conversely, GPx activity presented a negative correlation with FT4 and free triiodothyronine (FT3) levels (r = –0.934, p = 0.001; r = –0.993, p = 0.000, respectively). In addition, GPx activity showed positive correlation with selenium level (r = 0.981, p = 0.019) and the FT3 level correlated negatively with the level of thiol groups (r = –0.892, p = 0.017).

Conclusions:

This study shows the presence of an oxidative stress and selenium deficiency in HTX patients and suggests that the hyperthyroid state is strongly implicated in the establishment of this disturbed oxidative profile.

Keywords

antioxidant enzymes carbonyl hashitoxicosis selenium malondialdehyde

Introduction

Autoimmune thyroid diseases are common autoimmune pathologies affecting the thyroid gland [Eschler et al. 2011]. This group of diseases includes Hashimoto’s thyroiditis (HT) and Graves’ disease (GD) [Guarino et al. 2009]. The hallmark of these autoimmune thyroid pathologies is the presence of autoantibodies to various enzymes and proteins in the thyroid gland such as thyroid peroxidase (TPO), thyroglobulin (Tg) and thyroid-stimulating hormone receptor (TSHR) [Michels and Eisenbarth, 2010; Girgis et al. 2011]. Even though both HT and GD manifest infiltration of the thyroid with thyroid reactive lymphocytes, the end result is two clinically opposing syndromes. GD manifests by hyperthyroidism associated with an increase in free thyroxine (FT4), whereas HT is characterized by hypothyroidism which is generally due to low levels of FT4 [Girgis et al. 2011; Eschler et al. 2011; Bahn et al. 2011, Stuart et al. 2012].

However, HT begins with a transient hyperthyroid phase due to inflammation and destruction of thyroid cells causing release of thyroid-stimulating hormone (TSH) into the blood circulation [Johnstone et al. 2004; Unnikrishnan, 2014; Leyhe and Müssig, 2014; Harsch et al. 2008]. This clinical feature is known as hashitoxicosis (HTX) [Leyhe and Müssig 2014]. In fact, HTX initially described by Fatourechi and colleagues in 1971, has the clinical features of Graves’ hyperthyroidism and the pathological appearance of HT [Fatourechi et al. 1971]. Indeed, the serum TSH is suppressed and total FT3 and FT4 are elevated, but after a period of 3–24 months HTX evolves into permanent hypothyroidism [Leyhe and Müssig, 2014; Caturegli et al. 2014; Wasniewska et al. 2012]. Nevertheless, clinical variability and potential diagnostic pitfalls are sometimes encountered which makes the diagnosis of HTX quite difficult [Nabhan et al. 2005].

Autoimmune thyroid pathologies are well known to be associated with oxidative stress [Duracková, 2010; Hultqvist et al. 2009]. Oxidative stress is defined as an imbalance between the production of reactive oxygen species (ROS) and reactive metabolites, and their elimination by protective mechanisms comprising an enzymatic defense system which include superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) [Lassoued et al. 2010; Valko et al. 2007]. It is acknowledged that oxidative stress is implicated in several human pathologies including thyroid dysfunctions [Lassoued et al. 2010; Ben Mansour et al. 2008, 2010; Gargouri et al. 2009]. However, the link between HTX and oxidative stress has not yet been explored. The aim of this study was to evaluate the oxidative profile of HTX patients compared with healthy subjects. Several markers are considered: lipid peroxidation; oxidized proteins; and anti-oxidant enzymatic activities.

Patients and methods

Patients

Between April 2007 and April 2008, five newly diagnosed HTX Tunisian patients (one male and four females) were recruited from the Department of Endocrinology and Diabetes at Hedi Chaker University (Sfax, Tunisia) and the Department of Nuclear Medicine, CHU Habib Bourguiba, Sfax, Tunisia. The age of patients (mean ± standard deviation) was 51.25 ± 18.39 years old. The small sample size was due essentially to the difficulty of the diagnostic of the HTX pathology since it is a transitional phase whereby HT disease begins and usually passes as inconspicuous. The diagnostic of HTX was based on the presence of biochemical hyperthyroidism, as indicated by a decrease in TSH, an increase in FT4 level in association with the absence of antibodies toward TSHR and a high level of antibodies toward thyroid peroxidase (TPO) (Table 1).

Table 1.

Thyroid hormones and anti-thyroid autoantibodies levels in the plasma of HTX patients and controls. The results are expressed as mean ± SE.

Markers	Controls	HTX patients
FT4 (pg/ml)	9.44 ± 0.45	51.42 ± 7.59
FT3 (pg/ml)	2.70 ± 0.04	11.62 ± 5.4
TSH (µUI/ml)	1.38 ± 0.55	0.22 ± 0.0.03
Anti-TPO (UI/ml)	103.08 ± 8.10	446.26 ± 120.53
Anti-Tg (UI/ml)	43 ± 15.49	193.3 ± 30.82

FT, free thyroxine; HTX, hashitoxicosis; SE, standard error; Tg, thyroglobulin; TPO, thyroid peroxidase; TSH, thyroid-stimulating hormone.

All patients had no treatment for hyperthyroidism. The diagnosis was confirmed by spontaneous resolution of the hyperthyroidism with subsequent development of hypothyroidism. A total of 11 healthy individuals (2 males and 9 females) aged 45.31 ± 9.67 and who had no family history of autoimmune disease were used as controls. Patients and controls with diabetes mellitus, hypertension and undergoing hormonal therapy were excluded. Venous blood samples from patients and controls were centrifuged at 1500g for 10 min and the plasma separated. All samples were immediately frozen and stored at -80°C until analysis.

The study plan was approved by our institutional ethics committee, and informed consent was obtained for all patients and control subjects.

Hormonal and immunological thyroid tests

The concentration of TSH and the levels of the thyroid hormones FT4 and FT3 were determined using commercial kits (RIA-gnost® FT4 and FT3; CIS Bio, France; Vidas TSH, Biomerieux, France). The levels of anti-TPO and anti-Tg antibodies were determined by enzyme-linked immunosorbant assay (ELISA) using a commercial kit (Orgentec, Germany).

Lipid peroxidation

Lipid peroxidation was evaluated by the thiobarbituric acid reactive species (TBARS) method in the plasma of patients and controls [Buege and Aust, 1978].

For conjugated dienes, 25 µl of plasma were added to 3 ml of a solution of chloroform/methanol and then centrifuged for 5 min at 3000 revolutions per minute (rpm). A volume of 2 ml of the supernatant was dried at 45°C overnight. The extract obtained was dissolved in 2 ml of methanol and the optical density (OD) reading was performed at 233 nm [Gargouri et al. 2011].

Protein modification markers

Free thiol groups

Thiol level was determined based on the method of Hu [Hu, 1994]; 50µl of plasma were added to 1200 µl of phosphate buffer 0.05 M pH 8 and the first OD (OD1) was measured at 412 nm. After addition of 250µl of 5,5′-dithiobis 2-nitrobenzoate (DTNB) 10 mM and reposing for 15 min protected from light, the second OD (OD2) was measured at 412 nm. Values were reported to a calibration curve of N-acetylcysteine [Hu, 1994].

Malondialdehyde bound to proteins

For the determination of malondialdehyde (MDA) bound to plasma proteins, 200 µl of plasma were added to 800 µl of sulfuric acid (N/12) and of 100 µl of phosphotungstic acid (10%). After incubation for 5 min and centrifugation at 3000 rpm for 10 min, the supernatant was removed and the precipitated proteins were diluted in 800 µl of sterile water. Then 10 µl of ethylene diamine tetra acetic acid (EDTA) (0.14 mM), 5 µl of butylated hydroxytoluene (BHT) and 200 µl thiobarbituric acid (TBA) (1%) were added to the dilution and incubated at 95°C for 30 min. Finally, 600 µl of the reaction medium was mixed with 600 µl butanol. After centrifugation at 3000 rpm for 15 min, the OD in the butanol phase was at 532 nm. Results are expressed in nmol/g of total protein [Ben Mansour et al. 2010]

Protein carbonyl groups

The protein-bound carbonyl concentration was measured as described previously [Flohe and Gunzler, 1984]. The method was based on the reaction of the carbonyl group with 2,4-dinitrophenylhydrazine (DNPH) to give the corresponding hydrazone. This product can be quantified spectrophotometrically at 370 nm [Hawkins et al. 2009].

Antioxidant enzyme activities

SOD activity

SOD activity was determined by spectrophotometry (420 nm) using a pyrogallol assay modified as follows. The rate of pyrogallol autoxidation in tris-cacodylic acid diethylenetriaminepenta-acetic acid buffer (pH 8–8.2) was determined (A₁). The autoxidation of pyrogallol was evaluated under the same conditions after addition of 25 µl of plasma (A₂) [Ben Mansour et al. 2008]. The inhibition percentage of pyrogallol oxidation was determined using the following formula:

% i n h i b i t i o n = [\frac{(A 1 - A 2)}{A 1}] \times 100

CAT activity

The evaluation of CAT activity is based on the principle that the absorbance at 240 nm decreases because of hydrogen peroxide (H₂O₂) dismutation. The extinction coefficient 43.6 per mol per cm for H₂O₂ was used for the calculation. One unit was defined as the amount of H₂O₂ converted into H₂O and ½O₂ in 1 min under standard conditions, and the specific activity was reported as units per milligram of protein [Ben Mansour et al. 2008].

GPx activity

GPx activity was performed according to the method of Flohe and Gunzler which is based on glutathione oxidation by GPx in the presence of DTNB. The absorbance was determined at 412 nm. GPx activity was expressed as µmol of disappeared GSH/min/mg of proteins [Flohe and Gunzler, 1984].

Evaluation of selenium level

Selenium levels were determined by atomic absorption spectrophotometry [Ducros and Favier, 1992].

Protein quantification

A protein assay kit from Bio-Rad (France) was used to determine the protein level in the plasma of patients and controls. Bovine serum albumin served as a standard.

Statistical analyses

The statistical analysis was carried out using the Student’s t-test to assess the statistical significance of the differences between patients and controls. All data are expressed as mean ± standard error (SE). Correlation studies between markers were performed using Pearson correlation test. The level of significance was taken as p < 0.05. All statistical analyses were performed using IBM SPSS Statistics 20.

Results

Lipid peroxidation

Two markers are considered in the investigation of the lipid peroxidation. The TBARS level was evaluated by the TBARS method and the level of conjugated dienes by the spectrophotometric method described above. HTX patients showed an increase in levels of plasma TBARS and conjugated dienes compared with controls (p = 0.034 and p = 0.043, respectively) (Table 2).

Table 2.

Lipid peroxidation and antioxidant enzyme activities in the plasma of HTX patients and controls.

Markers	Controls	HTX patients	p value
MDA	0.070 ± 0.01	0.21 ± 0.075*	0.034
Conjugated dienes	0.115 ± 0.002	0.159 ± 0.022*	0.043
SOD	0.200 ± 0.026	0.396 ± 0.031***	0.000
CAT	0.179 ± 0.062	0.444 ± 0.055*	0.014
GPx	6072.193 ± 983.734	1214.469 ± 254.04***	0.000

Results are expressed as mean ± SE.

Statistical significance (Student’s t-test): *p < 0.05;**p < 0.01; ***p < 0.001.

CAT, catalase; GPx, glutathione peroxidase; HTX, hashitoxicosis; MDA, malondialdehyde; SE, standard error; SOD, superoxide dismutase.

Protein modification

For protein modification markers, carbonyls level as well as the level of MDA bound to proteins showed a significant increase compared with the control group (p = 0.000) (Figures 1 and 2). However, the thiol level presented a significant decrease compared with controls (p = 0.008) (Figure 3). These results confirmed the presence of protein oxidation in the plasma of HTX patients.

Figure 1.

Carbonyl level determination in HTX compared with a group of controls.

Figure 2.

MDA bound to proteins level determination in HTX compared with a group of controls.

Figure 3.

Thiol level determination in HTX compared with a group of controls.

Antioxidant enzyme activities

Antioxidant enzymes activities were evaluated using spectrophotometric methods. Our results showed that SOD and CAT activities are significantly increased whereas GPx activity decreased in comparison with healthy controls (p = 0.000; p = 0.014 and p = 0.000 respectively) (Table 2).

Plasmatic selenium level

The selenium level was assessed via atomic absorption in the plasma of patients and showed a significant decrease in HTX patients compared with healthy controls (p = 0.029) (Figure 4).

Figure 4.

Selenium level determination in HTX compared with a group of controls.

Correlation study

In HTX patients, a significant positive correlation was observed between TBARS and FT4 levels (r = 0.711, p = 0.048). In addition, FT4 level correlated positively with SOD activity (r = 0.713, p = 0.047). GPx activity showed a negative correlation with FT4 and FT3 levels (r = –0.934, p = 0.001; r = –0.993, p = 0.000, respectively). In addition, a positive and significant correlation was detected between GPx activity and selenium level (r = 0.981, p = 0.019). Moreover a significant negative correlation was also detected between FT3 and thiol levels (r = –0.892, p = 0.017). However, the FT4 level presented a positive correlation with the level of MDA bound to protein but without significance (r = 0.695, p = 0.056).

Discussion

Oxidative stress arises when the production of ROS exceeds cellular ability to remove them and repair cellular damage, and ultimately leads to the widespread oxidative damage to macromolecules, including lipid, proteins and DNA [Tsai et al. 2009, Marcocci et al. 2012]. Oxidative stress appears to be involved in the development of several diseases such as endocrine and inflammatory diseases [Tsai et al. 2009]. Moreover, several studies have reported that the oxidative stress was associated with autoimmune thyroid diseases such as GD and HT [Lassoued et al. 2010; Aslan et al. 2011; Žarković, 2012].

The present study is the first attempt to investigate oxidative stress in HTX patients. The oxidative profile was evidenced by the high rate of lipid peroxidation markers, protein modifications and disturbed antioxidant enzymatic activities in the plasma of HTX patients compared with healthy controls. Our results concerning lipid peroxidation are comparable with previous reports showing high levels of TBARS (endproducts of ROS degradation of polyunsaturated lipids) and conjugated dienes in the blood and in the thyroid tissues of GD patients compared with controls [Marcocci et al. 2012; Žarković, 2012; Ademoglu et al. 2006].

In addition to their involvement in lipid peroxidation, ROS can modify both the structure and the function of proteins. Metal-catalyzed protein oxidation results in the addition of carbonyl groups, cross-linking or fragmentation of proteins. Lipid (peroxidation) aldehydes can react with sulfhydryl (cysteine) or basic amino acids (histidine, lysine) [Shah et al. 2014]. The measurement of modified proteins in the plasma of HTX patients revealed a significant increase in the levels of the MDA–protein adducts and carbonyl groups, as well as a decrease in the concentration of thiol groups compared with healthy controls. These results are in accordance with those of other autoimmune diseases showing a diminution in the level of thiol groups and an increase in the level of MDA bound to protein in sera of patients suffering from other autoimmune pathologies [Ben Mansour et al. 2010; D’souza et al. 2008]. The proteins modified by oxidative stress can be neo-antigens for the immune response, as has been described in several other autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis [Kurien and Scofield, 2003].

The manifestation of oxidative damage will depend on the overall efficiency of the antioxidant mechanisms and their ability to cope with higher demands [Villanueva et al. 2013]. Antioxidant enzymes activities investigation in HTX patients showed an increase in SOD and CAT activities in comparison with healthy subjects. These high activities are generally explained as an adaptation to an increase in the oxidative stress. SOD and CAT usually act in a synergetic manner. SOD is one o f a group of metalloenzymes that scavenge superoxide radicals, dismutating O^2- into O₂ and H₂O₂, while CAT converts H₂O₂ to O₂ and H₂O [Ben Mansour et al. 2010; Žarković, 2012]. However, the activity of the seleno-enzyme GPx decreased in HTX patients compared with controls. This finding is similar to that of Bednarek and colleagues who noted a decrease in GPx and glutathione reductase activities in GD patients with and without infiltrative ophthalmopathy [Bednarek et al. 2005].

The decrease in the activity of GPx prompted us to evaluate the selenium level in the plasma of HTX patients. Interestingly, our results showed a decrease in the selenium plasma concentration compared with controls. This finding is in agreement with a previous study conducted on newly diagnosed GD and autoimmune hypothyroidism showing lower levels of serum selenium [Jain, 2014]. In addition, the positive correlation found between selenium concentration and GPx activity supports the hypothesis that the deficiency in selenium level may be the cause of the decrease in GPx activity. Indeed, this enzyme is the most popular selenoprotein and is a powerful antioxidant enzyme which mitigates the effects of oxidative stress by elimination of ROS [Dharmasena, 2014]. Besides, selenium deficiency leads to the accumulation of H₂O₂ and hydroperoxides (R–OOH) causing tissue inflammation and disease [Dharmasena, 2014; Thérond and Denis, 2005]. In addition to the decrease in selenium level, other factors causing decreased GPx activity should not be excluded such as the decrease in the level of thiols, which act as an electron donor and support an antioxidant role for glutathione peroxidase 3 (GPx3) in plasma [Brown and Arthur, 2001].

The major factor implicated in the establishment of the oxidative stress associated with HTX seems to be the high level of thyroid hormones. Several studies have shown that FT3 and FT4 hypersecretion is implicated in the establishment of the oxidative stress during hyperthyroidism [Lassoued et al. 2010; Fernandez et al. 2006]. Remarkably, our statistical studies showed a positive correlation between FT4 and TBARS levels, SOD activity and the level of MDA–protein adducts, while GPx activity showed a negative and significant correlation with FT4 and FT3 levels. These results support the implication of thyroid hormone hypersecretion associated with hyperthyroidism in the induction of the oxidative stress in HTX patients. Indeed, the hyperthyroidism which characterizes HTX as well as GD pathologies is well associated with increased oxygen consumption, dysfunction in the mitochondrial respiratory chain, elevated intracellular adenosine triphosphate (ATP) consumption, and increased ROS production – especially the superoxide (O₂^°-) [Marcocci et al. 2012]. This radical can lead to the formation of many other reactive species, including hydroxyl radicals (OH^°), which can readily start the free radical process of lipid peroxidation [Shah et al. 2014]. Nevertheless, these results do not exclude other potential sources of oxidative stress shown in HTX such as the strong inflammatory reaction accompanying the destruction of the thyroid gland, which could be responsible for this situation [Mayer et al. 2004].

Conclusion

This study has shown the presence of an oxidative stress in HTX patients characterized by lipid peroxidation, disturbed antioxidant enzymes activities and protein modifications in comparison with healthy controls. Furthermore, the reduced concentrations of selenium and thiols in the plasma of HTX patients may be the cause of reduced GPx activity. The oxidative stress found in HTX patients was probably due to the hyperthyroidism associated with this autoimmune thyroid pathology and the selenium deficiency. Selenium administration could insure a better recovery from autoimmune thyroid disease.

Footnotes

Acknowledgements

Special thanks are due to Dr François Laporte for his help with selenium dosage.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported financially by the Ministry of Higher Education, Scientific Research and Information and Communication Technologies, Tunisia.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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Lipid peroxidation,proteins modifications,anti-oxidant enzymes activities and selenium deficiency in the plasma of hashitoxicosis patients

Abstract

Objectives:

Patients and methods:

Results:

Conclusions:

Keywords

Introduction

Patients and methods

Patients

Lipid peroxidation

Protein modification markers

Free thiol groups

Malondialdehyde bound to proteins

Protein carbonyl groups

Antioxidant enzyme activities

SOD activity

CAT activity

GPx activity

Evaluation of selenium level

Protein quantification

Statistical analyses

Results

Lipid peroxidation

Protein modification

Antioxidant enzyme activities

Plasmatic selenium level

Correlation study

Discussion

Conclusion

Footnotes

Acknowledgements

Funding

Declaration of Conflicting Interests

References