Autoimmune diseases are characterized by body's immune responses targeting
its own tissues, causing prolonged inflammation and subsequent tissue destruction.
A healthy immune system recognizes, identifies, remembers, attacks and destroys
foreign antigens and cancer cells. A defective immune system, on the other hand,
inflicts chaos in host body by directing antibodies against its own tissues.
Any disease in which cytotoxic cells are directed against self-antigens in the
body's tissues is considered autoimmune in nature. Hashimotos thyroiditis
which is an autoimmune thyroid disease, is one of the most frequent causes of
hypothyroidism which is among the most common endocrine disorders in India (Kochupillai,
Oxidative Stress (OS) has been implicated in a number of diseases such as cardiovascular
diseases, renal diseases, diabetes, inflammatory problems etc. (Krinsky,
1992). In the previous study, we found increased level of OS in untreated
hypothyroid patients (Nanda et al., 2007, 2008).
Hashimotos thyroiditis which is a common cause of hypothyroidism, is characterized
by elevated Anti-thyroperoxidase Antibody (TPO Ab). There exists a good correlation
between the degree of lymphocytic infiltration of the thyroid gland and the
presence of TPO antibodies (Portmann et al., 1985).
Association of oxidative stress with various autoimmune diseases has been reported
(Kumagai et al., 2003; Kumagai,
2003). However, the association of thyroid antibodies and OS in the pathophysiology
of hypothyroidism is not fully understood. Hence, in the present study, we have
analyzed the link between anti TPO antibodies and OS in hypothyroid patients.
MATERIALS AND METHODS
Subjects: The present study comprises of sixty seven (55 females and 12 males) newly diagnosed patients with primary hypothyroidism (TSH level more than 10 μIU mL-1). They were recruited consecutively from the out patient department of medicine, Jawaharlal Institute of Post-graduate Medical Education and Research (JIPMER) in the study group. Subjects receiving lipid lowering drugs or antioxidant vitamin supplements, pregnant women, women on hormone replacement therapy, alcoholics, smokers, patients with hypertension and patients suffering from diseases other than hypothyroidism were excluded from the study groups. This study was approved by the research council and human ethics committee of JIPMER. Written consent was obtained from all the subjects. The patients were enrolled for the study prior to initiation of therapy.
Blood collection: Overnight fasting blood samples (serum and EDTA whole blood) were collected in separate tubes. EDTA whole blood was immediately used for the estimation of whole blood reduced glutathione. Serum was used for the estimations of glucose and lipid profile on the same day. Remaining serum samples were refrigerated at-50°C till the estimations of thyroid profile, Protein Carbonyls (PCO), Malondialdehyde (MDA) as thiobarbituric acid reactive substance and usCRP were carried out.
Thyroid profile: Total Tri-iodothyronine (T3) and total tetra-iodothyronine (T4) were estimated by RIA and TSH was estimated by IRMA using kits procured from BARC (Bhaba Atomic Research Center, Mumbai, India).
Lipid profile: Estimation of serum lipid profile was carried out by
using various commercial kits. Total Cholesterol (TC) and Triglycerides (TG)
were estimated by enzymatic methods using kits from Biocon (Germany) and Accurex
(Mumbai, India), respectively. LDL and Very Low Density Lipoprotein Cholesterol
(VLDL C) were precipitated using the phosphor-tungstate magnesium acetate reagent
from Agappe diagnostics (Thane, India). HDLC was estimated in the supernatant
while LDL C was calculated using the Fridewald formula (Friedewald
et al., 1972).
Glucose: Serum glucose was estimated using glucose oxidase kits obtained from Dr. Reddys lab (Hyderabad, India) using an autoanalyzer (550 express plus, Bayers Diagnostics, USA).
Antioxidants: Activity of antioxidants such as reduced glutathione
(GSH), Glutathione Peroxidase (GPx), Glutathione S Transferase (GST) and Catalase
were measured by standard methods described previously (Nanda
et al., 2008).
Lipid peroxide and protein carbonyl: Malondialdehyde, the end product
of lipid peroxidation was estimated by thiobarbituric acid method modified by
K Satoh (Satoh, 1978) and Protein Carbonyl (PCO) was
estimated by the method of Levine (Levine et al.,
Anti-thyroperoxidase antibody and us CRP: Anti-thyroperoxidase Antibody (TPO) titer was estimated by ELISA kit (Varelisa, Pharmacia and Upjohn, Germany) following the manufacturers instruction. It is based on noncompetitive enzyme immuno assay for the quantitative estimation of TPO. The cut off for positive TPO antibody titer is 100.
Quantitative assay of ultrasensitive CRP was performed by a turbidimetric immunoassay kit adapted to autoanalyser (Aptec Diagnostics, Belgium). The ultrasensitive assay for CRP (usCRP) is analogous to high sensitive CRP (hsCRP) assay.
Statistical analyses: All parameters were expressed as Mean±SD. Significance of the differences between control and test groups were analyzed by students t test. For parameters without normal distribution, we used Mann Whitney-U test for analyzing the significance of differences. The correlations were assessed by Pearson correlation and shown in Table 2. All the statistical analyses were performed using the SPSS software (SPSS Inc, Chicago, USA).
RESULTS AND DISCUSSION
In present study, out of sixty seven cases, thirty nine were TPO positive and twenty eight were TPO negative. When compared between TPO positive and TPO negative cases, MDA was fond significantly higher in the former group indicating increased OS in autoimmune cases of hypothyroidism (Table 1). There was no difference in the level of protein carbonyls, antioxidants and us CRP between the two groups.
||Comparison of the anthropometric and biochemical and oxidative
stress parameters between anti-TPO antibody positive and anti-TPO antibody
negative cases of untreated hypothyroidism
|All parameters analysed by students unpaired student
t test, except TSH and anti-TPO Ab which were analysed by Mann
whitney U test; *p<0.05 and ### p<0.001
|| Pearson correlation of anti-TPO antibody with various parameters
in TPO positive cases of hypothyroid patients (n = 39)
Protein carbonylation is an important biochemical modification which affects
the structure and function of proteins. The bodys antioxidant defense
system protects against such derangements. Autoimmune response is closely associated
with the generation of free radicals (Kurien and Scofield,
Altered antioxidant status was reported in Hashimoto cases in only one previous
study (Gerenova and Gadjeva, 2007) in comparison to
healthy controls. However, they did not compare the OS level between TPO Ab
positive and negative cases. In the present study, we found significantly higher
OS in autoimmune hypothyroidism compared to non autoimmune hypothyroidism (Table
1). To the best of our knowledge this comparison has not been reported by
any other study till date. In previous studies, patients with various thyroid
functional states of autoimmune diseases concluded that oxidative stress occurs
at ATD in spite of their altered thyroid status (Tsotsonava
et al., 2007a, b). In these two studies,
only 10 and 19 patients with autoimmune hypothyroidism were included respectively.
They found a positive correlation between lipid peroxy radicals and FT4 and
advocated inclusion of antioxidants for chronic autoimmune thyroiditis cases.
Our present findings corroborates with these reports. In this study, the sample
size is larger than the previous reports (39 cases). In this study, there was
a direct correlation of TPO Ab titer with various parameters of OS and inflammation
which was not reported earlier.
However, the marginal difference in MDA in TPO Ab positive cases compared to non autoimmune hypothyroidism may indicate that the contribution of autoimmunity towards the genesis of OS might not be substantial. Nevertheless, increase in anti TPO Ab titer significantly correlated with the advancement of age and decreased levels of T4, oxidative stress parameters such as MDA, PCO and increase in inflammatory markers like usCRP. Hence, the association of autoimmunity and OS in the pathophysiology and complications of hypothyroidism could be substantial in the long run.
Absence of significant correlation of anti-TPO Ab with TSH may indicate that degree of autoimmunity is not directly linked to the severity of the dysfunction. OS is known to increase with age. Also, incidence of hypothyroidism increases with age. As in the present study, anti-TPO Ab was positively correlated with age, we presume that presence of TPO antibody especially in aged hypothyroid patients could be an important factor in accelerated OS in hypothyroidism. The association of TPO Ab with serum us-CRP, MDA and PCO suggests that inflammation and OS are exacerbated in TPO positive hypothyroid cases due to additional autoimmune assaults.
Hypothyroidism is a treatable endocrine dysfunction. Hence, most often its
potential complications are not given due attention. However, it is a disease
that requires life long drug supplementation with careful monitoring of thyroid
profile to avoid iatrogenic side effects especially on heart. Often the drug
therapy takes long to normalize the thyroid profile (Libby,
2005; Mishkel and Crowther, 1977). Flynn
et al. (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 and cerebrovascular diseases.
Hence, from the present study, keeping in view the increase in OS parameters,
judicious inclusion of antioxidant therapy may be recommended to hypothyroid
patients especially in anti TPO Ab positive hypothyroid patients. In the later
group of patients the antioxidant treatment may be continued even after attainment
of euthyroidism till significant decrease in their TPO in titer.
We acknowledge the research grant from ICMR (Indian Council of Medical Research) in the form of adhoc research grant to Dr. Zachariah Bobby and Senior research fellowship to Dr. Nivedita Nanda. We also acknowledge the techniqual assistance of Mr. R. Vengattraman for this study.