Arch. Pharm. Res. DOI 10.1007/s12272-013-0227-y
RESEARCH ARTICLE
The effect of levothyroxine replacement therapy on lipid profile and oxidative stress parameters in patients with subclinical hypothyroid Serkan Mutlu • Adem Parlak • Umit Aydogan • Aydogan Aydogdu • Bugra Soykut • Cemal Akay • Kenan Saglam • Abdullah Taslipinar
Received: 15 January 2013 / Accepted: 25 July 2013 Ó The Pharmaceutical Society of Korea 2013
Abstract The purpose of this present study is to investigate the levels of oxidative stress parameters in patients with subclinical hypothyroidism (SH) and the effects of levothyroxine (LT4) replacement therapy on these parameters and lipid profile. At the beginning of the study blood samples were collected from the patients in order to analyse oxidative stress parameters, lipid profile and biochemical markers. After replacement therapy with LT4, in the third month, same tests were performed again. At the baseline superoxide dismutase (SOD) levels were found to be higher in SH patients, compared to the euthyroid group. After LT4 therapy, statistically significant decreases in SOD and catalase levels and increase in HDL-C levels were noticed. LT4 treatment was found to have positive effects on oxidative stress indicators and HDL-C levels.
This work has never been submitted for publication previously and not been submitted for publication elsewhere. All authors have reviewed and approved this manuscript for publication. S. Mutlu (&) U. Aydogan Department of Family Medicine, Gulhane School of Medicine, Ankara, Turkey e-mail:
[email protected] U. Aydogan e-mail:
[email protected] A. Parlak Department of Family Medicine, Ag˘rı Military Hospital, Agri, Turkey e-mail:
[email protected] A. Aydogdu A. Taslipinar Department of Endocrinology and Metabolic Diseases, Gulhane School of Medicine, Ankara, Turkey e-mail:
[email protected]
Keywords Subclinical hypothyroidism Oxidative stress SOD CAT Lipid Levothyroxine
Introduction Subclinical hypothyroidism (SH) is a common thyroid disease defined as elevated thyroid stimulating hormone (TSH) levels while the free T3 (FT3) and free T4 (FT4) levels are in the reference range (Canaris et al. 2000; Zulewski et al. 1997). Under normal physiological conditions, thyroid hormones are the most significant factors having an effect on the basal metabolic rate by modifying the main production site of the free radicals and changing the mitochondrial oxygen consumption. Therefore, the fact that the changes in the thyroid hormone levels affect the mitochondrial free radical production is not surprising. It is shown that the thyroid hormones affect the synthesis and reduction of enzymes, vitamins and antioxidant proteins (Oziol et al. 2003; Freeman and Crapo 1982; Pereira et al. 1994). A. Taslipinar e-mail:
[email protected] B. Soykut C. Akay Department of Pharmaceutical Toxicology, Gulhane School of Medicine, Ankara, Turkey e-mail:
[email protected] C. Akay e-mail:
[email protected] K. Saglam Department of Internal Medicine, Gulhane School of Medicine, Ankara, Turkey e-mail:
[email protected]
123
S. Mutlu et al.
Organisms developed defence systems called antioxidants in order to neutralize the toxic free radicals. The molecules acting in this system play a role in cleaning the free oxygen radicals and consequently preventing oxidative damage. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S transferase (GST) are among the antioxidant enzymes that are primary in the enzymatic system having cellular-level effect. It is possible to determine the oxidative damage by measuring the levels of these molecules (Halliwell 1996, 2012). Serum lipid and lipoprotein concentrations generally change in thyroid disorders. Increase in total cholesterol (TC), low-density lipoprotein (LDL-C) and triglyceride (TG) levels along with increased TSH and decrease in high-density lipoprotein (HDL-C) levels are observed even in normal range TSH levels (Asvold et al. 2007). Thyroid function disorder is a metabolic condition that affects many metabolic body conditions. Although there are many researches about how oxidative stress and cholesterol metabolism is affected in obvious hypothyroidism, conflicting results have been reported about how the body change in SH cases. Also, how thyroid replacement therapy will affect oxidative stress in SH patients arouses curiosity. That is why in the present study, levels of oxidative stress parameters in patients with SH and the effect of levothyroxine (LT4) therapy on oxidative stress in patients with SH is investigated based on SOD and CAT levels. Furthermore, the change in the lipid profile with LT4 therapy is investigated as well.
Materials and methods Thirty caucasian patients (18 females, 12 males) recently diagnosed with SH applied to Gulhane School of Medicine Endocrinology and Internal Medicine outpatient clinics between 2011 January and 2012 June and 30 healthy volunteers as control group (18 females, 12 males) were included in the study. Study inclusion criteria 1. 2. 3. 4. 5.
Patients with normal serum FT4 and FT3 levels, high serum TSH level Patients aged 18–55 Patients with no mental or cognitive function disorder Patients with no autoimmune history Patients with no acute infection
Study exclusion criteria 1. Hypertensive patients 2. Patients with diabetes mellitus
123
3. 4. 5. 6. 7. 8.
9. 10.
Patients receiving radioactive iodine treatment Patients with hepatic lipidosis Patients with dyslipidemia and/or taking lipid-lowering medication Patients diagnosed with a thyroid disease other than and taking thyroid medication Smoking patients Patients taking non-steroidal anti-inflammatory medication, vitamin A, vitamin C and vitamin E for the past 3 months Patients diagnosed with chronic inflammatory diseases and/or taking medication for such diseases Patients with thyroidectomy
All patients are informed of the study and respective consents are taken. The study is approved by the ethics board of ‘Gulhane School of Medicine Presidency of Local Ethics Board’ dated 13.10.2010 and protocol number 1491-1061-10/1539. Follow-up The patients were informed of their disease and routine examinations were performed, and then recorded in the patient follow-up form with predefined socio-demographic data such as age, height, weight and body mass index (BMI). After minimum 8 h of fasting, venous blood samples were collected from patients for fasting blood glucose (FBG), urea, creatinine, TC, TG, HDL-C, LDL-C, aspartate aminotransferase (AST), alanine aminotransferase (ALT), FT3, FT4, TSH, biochemical parameters as well as SOD and CAT level determination at 8.00 am. Thyroid ultrasound (USG) examinations of the patients were also planned at the time of diagnosis and probable nodule existence in thyroid tissues as well as whether the current condition is caused by Hashimoto disease or not is investigated. 25–50 lg/day oral LT4 sodium treatment was started to SH patients. Since the aim is to achieve euthyroidism in the therapy group, TSH levels were checked at the end of the first month of the therapy and the dosage was adjusted in a way that the TSH level shall be below 5 lIU/mL. At the end of 3 months, blood samples were collected from patients with TSH values below the specified level in order to analyse the changes in lipid and oxidative stress levels.
Measurement of SOD and CAT parameters Biological analysis material collection from subjects Approximately 5 mL blood samples were collected in tubes with tri-potassium EDTA content in order to study
Levothyroxine therapy on oxidative stress
oxidative stress parameters. Accordingly the blood samples were immediately centrifuged at 4 °C and 4,400 rpm for 5 min. Plasma part were taken into polypropylene tubes and stored at -80 °C until analyzed. GBC Cintra 303 UV Spectrophotometry device (Serial Number: V3772) was used for measuring enzyme activities. Erythrocyte CuZn–SOD activity determination method In this method, superoxide radical generated by xanthine oxidase (XOD) system and the formed superoxide radical reacts with iodonitrotetrazolium (INT) and forms violet colored formazan dye. This formed color intensity was measured at 505 nm wavelength. Depending on the CuZn– SOD activity in the environment, this reaction was prevented and inhibition % was calculated. Inhibition % values obtained for the samples were also entered into this chart and their concentrations were calculated as kU/mL. Then, multiplied by the dilution factor 400 and the erythrocyte activity is calculated. Erythrocyte CAT activity determination method CAT converts hydrogen peroxide into water and molecular oxygen using catalytic activity. The principle of this method is based on CAT burning the present H2O2 time dependently and measurement of the reduced absorbance of hydrogen peroxide yielding maximum absorbency at 240 nm wavelengths. Decomposition rate of substrate H2O2 at 25 °C was observed at 240 nm for 30 s via spectrophotometric method. The calibration chart was prepared under the same conditions by using 0.01-0.035 kU/mL standard CAT solutions instead of erythrocyte hemolysate. The enzyme activities in the samples were calculated using chart. The activity was represented as kU/mL.
Statistical analysis The statistical evaluation of the data was performed using a microprocessor and commercial statistics software (SPSS ver. 15.0, SPSS Inc., Chicago, IL, USA). Primarily Kolmogorov–Smirnov goodness of fit test and normality analyses were taken into account for the analysis of continuous data. Parametric tests were used for the data conforming to normal distribution and non-parametric tests are used for the data not conforming to normal distribution. The differences between the groups were investigated using either Chi square or Student t tests as required by the number of groups and data distribution. The primary main outcome of the study were SOD and CAT, and the secondary main outcome were HDL, LDL, TC and TG. The
relationship between the investigated variables was evaluated using multivariate regression analysis. SOD, CAT, TSH, FT3, and FT4 were used as dependent variables and TG, TC, HDL, LDL, anti thyroid peroxidase (anti-TPO), anti thyroglobuline (anti-Tg), FBG, urea, and creatinine were used as independent variables in the multivariate regression analysis. SOD and CAT are also considered as independent variables among the data of which the effect on the thyroid hormone was evaluated. The data were calculated as mean ± standard deviation, number and percentage; alpha degree of freedom was determined as 0.05 in all tests and the value of calculated p being equal/ lower than 0.05 was considered as significant.
Results Socio-demographic characteristics, thyroid function tests, anti-TPO and anti-Tg antibody levels of both patient and control groups are shown in Table 1. A statistical difference between TSH levels of both patient and control groups were detected as expected (patient and control groups, respectively 8.30 ± 1.78 and 2.70 ± 0.99 lIU/mL, p \ 0.0001). No statistical difference between biochemical variables and lipid profiles of both patient and control groups were detected except for AST (for all parameters p [ 0.05, Table 1). The oxidative stress parameter SOD levels were detected as 669.89 ± 192.44 kU/mL in the patient group, and as 328.80 ± 52.51 kU/mL in the control group, so SOD levels were found to be significantly higher in the patient group (p \ 0.0001). No statistically significant difference was detected between the two groups in terms of CAT values evaluated as another oxidative stress parameter (patient and control groups, respectively 89.61 ± 27.03 and 95.91 ± 26.99 kU/mL, p [ 0.05). When patients with SH were compared with euthyroid healthy patients in terms of oxidative stress markers, statistically significant high levels of CAT were found in males (in SH group, CAT levels of females and males; respectively 81.43 ± 26.89 and 101.90 ± 23.12 kU/mL, p = 0.04; in euthyroid group, CAT levels of females and males; respectively 88.19 ± 27.03 and 107.49 ± 23.40 kU/mL, p = 0.05). It was detected that the thyroid hormones and the oxidative stress parameter SOD levels were affected by antiTPO and TSH levels (for anti-TPO; p \ 0.0001, beta = 0.55, 95 % CI 0.15–0.44 and for TSH; p \ 0.0001, beta = 0.61, 95 % CI 23.56–62.09). No similar relationship was detected for CAT levels (Table 2). It was observed in the multivariate regression analysis that the TSH values were affected by anti-TPO (p = 0.007, beta = 0.30, 95 % CI 0.001–0.004), anti-Tg (p = 0.03, beta = 0.18, 95 % CI 0.001–0.002) and SOD (p \ 0.0001,
123
S. Mutlu et al. Table 1 Comparison of patients with subclinical hypothyroidism with euthyroid healthy group based on socio-demographic, thyroid function test, biochemical analyses and oxidative stress parameters Parameters
Subclinical hypothyroid group (n = 30)
Euthyroid healthy volunteer group (n = 30)
p
Normal range
Age (years)
35.46 ± 8.16
34.33 ± 6.53
NSa
Female (%)
60
60
NSb
Height (m) Weight (kg)
1.67 ± 0.74 69.90 ± 13.19
1.68 ± 0.74 70.83 ± 11.12
NSa NSa
BMI (kg/m2)
24.81 ± 3.96
25.00 ± 3.27
NSa
18.5–24.9
FBG (mg/dL)
92.66 ± 9.34
91.46 ± 6.85
NSa
70–110
Urea (mg/dL)
25.40 ± 8.93
24.03 ± 7.14
NSa
10–50
Creatinine (mg/dL)
0.91 ± 0.16
0.89 ± 0.14
NSa
0.6–1.2
ALT (U/L)
16.90 ± 10.56
19.70 ± 9.28
NSa
10–40 a,*
AST (U/L)
17.96 ± 4.23
20.36 ± 3.61
p = 0.022
13–40
LDL (mg/dL)
126.86 ± 29.41
123.40 ± 35.62
NSa
50–130
HDL (mg/dL)
48.23 ± 7.66
52.13 ± 8.64
NSa
40–60
TC (mg/dL)
198.70 ± 34.83
188.60 ± 40.52
NSa
130–200
TG (mg/dL)
123.76 ± 55.28
111.83 ± 49.48
NSa
35–160
a
2.5–3.9
FT3 (pg/mL)
3.20 ± 0.58
3.07 ± 0.38
NS
FT4 (ng/dL)
1.09 ± 0.49
1.07 ± 0.18
NSa
0.61–1.12
TSH (lIU/mL)
8.30 ± 1.78
2.70 ± 0.99
p < 0.0001a,***
0.4–4.0
Anti-TPO (IU/lL) Anti-Tg (IU/mL)
563.16 ± 424.14 492.58 ± 993.17
1.09 ± 0.89 1.51 ± 1.10
p < 0.0001a,*** p = 0.009a,**
0–100 0–100
SOD (kU/mL)
669.89 ± 192.44
328.80 ± 52.51
p < 0.0001a,***
CAT (kU/mL)
89.61 ± 27.03
95.91 ± 26.99
NSa
Differences between euthyroid and subclinical hypothyroid groups are determined in the values of solely AST among biochemical tests; TSH, anti-TPO and anti-Tg among thyroid function tests; and the oxidative stress indicators SOD and CAT BMI body mass index, FBG fasting blood glucose, ALT alanine amino transferase, AST aspartate aminotransferase, LDL low-density lipoprotein, HDL high-density, TC total cholesterol, TG triglyceride, FT3 free T3, FT4 free T4, TSH thyroid stimulating hormone, anti-TPO anti thyroid peroxydase, anti-Tg anti thyroglobuline, SOD superoxide dismutase, CAT catalase, NS non-significant * p \ 0.05; ** p \ 0.01; *** p \ 0.001 a
Independent sample t test
b
Chi square, data are presented as mean ± standard deviation percentage
beta = 0.50, 95 % CI 0.01–0.04) levels. FT4 levels were affected by LDL levels (p = 0.04, beta = 0.61, 95 % CI 0.001–0.014). No similar relationship was detected for FT3 levels (Table 3). TSH levels reduced significantly after LT4 treatment in SH patients (8.30 ± 1.78 vs. 2.96 ± 1.00 lIU/mL, p \ 0.0001). Statistically significant change was observed in FT4 levels of SH patients after the LT4 treatment (preand post-therapy values, respectively 1.09 ± 0.49 and 1.24 ± 0.38 ng/dL, p = 0.024). After LT4 treatment, significant decreases were determined with the levels of SOD (737.55 ± 138.48 , 580.54 ± 196.82 kU/mL, p = 0.04) and CAT (101.90 ± 23.12 , 75.68 ± 35.69 kU/mL, p = 0.01) in male patients with SH but in female patients only CAT levels decreased after LT4 treatment (81.43 ± 26.89 , 54.10 ± 34.44 kU/mL, p = 0.02).
123
Statistically significant decrease was detected in the SOD values compared to the pre-LT4 therapy period (669.89 ± 192.44 vs. 564.40 ± 162.24 kU/mL, p = 0.019, Fig. 1). The CAT level being 89.61 ± 27.03 kU/mL in the pretherapy period was reduced to 62.73 ± 35.97 kU/mL level after LT4 treatment, and this reduction was considered as statistically significant (p = 0.001, Fig. 1). A statistically significant improvement was observed in the HDL values with LT4 therapy (pre- and post-therapy values respectively 48.23 ± 7.66 and 51.76 ± 7.94 mg/ dL, p = 0.004, Fig. 1). After LT4 replacement therapy statistically significant difference was observed in both male and female patients with SH (male; 44.00 ± 7.79 vs. 48.00 ± 6.28 mg/dL, p = 0.05, female; 51.05 ± 6.31 vs. 54.27 ± 8.08 mg/dL, p = 0.04). No statistical difference was observed in other lipid parameters.
** p \ 0.001
Discussion
* Multivariate regression analysis
95 % CI 95 % confidence interval, Independent variables: TG triglyceride, TC total cholesterol, HDL high-density lipoprotein, LDL low-density lipoprotein, Anti-TPO anti-thyroide peroxidase, anti-TG antithyroglobuline, TSH thyroid stimulating hormone, FT3 free T3, FT4 free T4, FBG fasting blood glucose, Dependent Variables: SOD superoxide dismutase, CAT catalase
When effects of biochemical parameters, thyroid antibodies, SOD and CAT levels on thyroid hormone levels were evaluated by multivariate regression analysis, it was observed that only anti-TPO and TSH levels found to affect SOD levels
-51.31–65.89 -1.13–0.81 -0.51–1.41 -19.27–24.91 -7.69–24.43 -3.93–1.97 -0.01–0.05 -0.03–0.01 -0.31–0.72 -0.97–1.31 -0.05–0.37 95 % CI
-0.94–0.07
0.80
0.04 -0.04
0.74 0.34
0.13 0.03
0.79 0.30
0.15 -0.11
0.50 0.38
-0.11 -0.15
0.34 0.41
0.25 0.05
0.76 0.09
0.31 Beta
-0.61
0.13 p* CAT
-367.62–550.73 -9.52–5.69 -7.54–7.70 -268.8–72.9 -100.9–153.63 23.56–62.09 -0.08–0.07 0.15–0.44 -5.14–2.98 -7.88–10.05 -1.08–2.32 95 % CI
-2.94–5.24
0.06
0.69 0.61
-0.06 0.003
0.98 0.25
-0.16 0.05
0.67
