Effects Of Levothyroxine Replacement Therapy On Serum Eicosanoids Metabolism In Subclinical Hypothyroidism Patients

Background:Subclinical hypothyroidism syndrome (subclinical hypothyroidism in abbreviation, SH) is a common endocrine disease, which is characterized by elevated level of serum thyroid stimulating hormone (TSH), but normal levels of serum free triiodothyronine (FT3) and serum free thyroxine (FT4). Subclinical hypothyroidism can be divided into mild subclinical hypothyroidism (4.2mIU/L＜TSH≤10mIU/L) and severe subclinical hypothyroidism (TSH＞10mIU/L) based on serum TSH level. Studies have shown that subclinical hypothyroidism is an independent risk factor for atherosclerosis and myocardial infarction, in addition to other factors such as LDL-C. And the morbidity and mortality of coronary heart disease in subclinical hypothyroidism patients increased with the elevating of TSH; Atherosclerosis pathogenetic conditions were significantly correlated with serum TSH levels. Due to subclinical hypothyroidism is closely related with the occurrence of a variety of serious diseases such as atherosclerosis, most scholars believe that, although subclinical hypothyroidism patients have no obvious clinical symptoms, they should be treated with thyroxine and other drugs as the replacement therapy for reducing the occurrence of subclinical hypothyroidism caused atherosclerosis, coronary heart disease and other serious diseases. However, currently as to when and how to give replacement therapy to subclinical hypothyroidism patients are still controversial, most scholars believe that subclinical hypothyroidism patients with serum TSH>10 mIU/L need replacement therapy, and these patients can benefit from replacement therapy; While whether subclinical hypothyroidism patients with a serum level of TSH≤10 mIU/LSH need replacement therapy, but if the replacement therapy can reduce the risk of atherosclerosis and ischemic heart disease, the conclusions were inconsistent presently.There are a lot of studies about the influence of levothyroxine replacement therapy on blood lipid characteristics of subclinical hypothyroidism patients, but which still are controversial. Some studies have shown a significant increase of serum PLA2-ⅡA (the key enzyme for phospholipid degradation) in subclinical hypothyroidism patients, and TSH can promote phospholipid degradation to produce a large amount of fatty acids including arachidonic acids. Arachidonic acid is a precursor of eicosanoids and an essential polyunsaturated fatty acids in mammals. Phospholipids hydrolysis, catalyzed by Ca2+ regulated cytosolic phospholipase A2 (cPLA2), is its important source. Catalized by the enzymes, arachidonic acid can generate a variety of eicosanoids with different biological activity through different metabolic pathways. As the key signaling molecules, these eicosanoids play important regulatory roles in inflammation, platelet function, coagulation, allergic reactions and oxidative stress. In particular, some eicosanoids play key roles in genesis and development of atherosclerosis (a chronic inflammatory disease), and are novel biomarkers of atherosclerosis. Therefore, it is necessary to reveal the eicosanoids dysbolism in subclinical hypothyroidism patients and the impact of levothyroxine replacement therapy on it. This will have important significance for understanding the relationship between subclinical hypothyroidism and cardiovascular disease, determining the timing of replacement therapy and evaluation of the significance of replacement therapy, and so on. However, studies on these issues have not yet been reported so far.Research purposes:1. To observe the effects of short-term levothyroxine replacement therapy on blood lipid levels and thyroid function in mild or severe subclinical hypothyroidism patients.2. To compare the changes of serum cPLA2 level, before and after the levothyroxine replacement treatment, between mild or severe subclinical hypothyroidism patients and the normal control group; To observe the influence of TSH on intracellular cPLA2 content, activity and expression level, and the relationship between extracellular cPLA2 level and intracellular cPLA2 activity; To discuss the influence of TSH on the key enzyme cPLA2 in the eicosanoids metabolism pathway.3. To compare the changes of major eicosanoids in different metabolic pathways before and after the levothyroxine replacement therapy in mild or severe subclinical hypothyroidism patients; explore the timing of the replacement therapy and evaluate the effects of the replacement therapy on eicosanoids metabolism.4. To analyze the intercommunication between eicosanoids level and a variety of factors, including TSH, and to understand the potential mechanisms of increased onset risk for atherosclerosis in subclinical hypothyroidism patients.Subjects:Subjects in this study were from the epidemiological survey population of Shandong province Ningyang County during January 2013 to February 2013, strictly followed the established inclusion and exclusion criteria. The results of two seprated tests were all in line with subclinical hypothyroidism and with a final diagnosis of subclinical hypothyroidism. Selected 30 cases of mild subclinical hypothyroidism patients (TSH 4.2-10.0mIU/L); 20 cases of severe subclinical hypothyroidism patients (TSH＞10mIU/L); and 22 cases of age, body mass index matched normal subjects. Mild subclinical hypothyroidism patients were treated with levothyroxine tablets taking 25 mcg daily by mouth, and severe subclinical hypothyroidism patients were treated with levothyroxine tablets taking 50 mcg daily by mouth, for 3 months.Research methods:1. Observating the effects of levothyroxine replacement therapy on thyroid function and blood lipids in subclinical hypothyroidism patients:collected clinical data of the subjects, recorded demographic characteristics of the included objects; Measured routine biochemical indicators, thyroid function, and blood lipids of blood samples in subclinical hypothyroidism patients before and after the treatment.2. Discussing the influence of TSH on cPLA2:serum cPLA2 level was determined by immunosorbent assay in mild or severe subclinical hypothyroidism patients before and after the treatment and compared. In vitro experiments, normal human peripheral blood mononuclear cells (PBMC) were treated with different concentrations of bovine TSH, relationships between the changes of intracellular and extracellularc PLA2 activities and contents with TSH were detected;3. To investigate the effects of levothyroxine replacement therapy on eicosanoids of different metabolic pathways in subclinical hypothyroidism patients:Thermo Vantage triple quadrupole mass spectrometer (LC-ESI-MS) was used to detect the changes of eicosanoids of different pathways in subclinical hypothyroidism patients before and after treatment; Serum 8-isoprostane (8-isoPG2a) level, the marker of oxidative stress, was evaluated by enzyme-linked immunosorbent assay before and after treatment.Statistical Analysis:In this study, the measurement data were described as mean ± standard deviation (x±s). Differences between groups were analyzed by ANOVA for the continuous variable data in line with the normal distribution, while analyzed by Kruskal-Wallis test for the continuous variable data in line with non-normal distribution. Correlation analysis among factors was conducted using Pearson’s correlation analysis and multiple regression analysis. Significance threshold was p＜0.05. Above analyses were finished with SPSS Statistics 18.0 (SPSS Inc., Chicago, IL, USA).Result:1. Compared with the normal control group, TC and TG of patients in mild subclinical hypothyroidism group and severe subclinical hypothyroidism group were increased but not significantly.Among thyroid-related hormones, FT3 and FT4 in all groups were not significantly different; But compared with the normal control group, serum TSH levels of patients in both mild subclinical hypothyroidism and severe subclinical hypothyroidism groups were significantly increased, and which were significantly higher in the severe subclinical hypothyroidism group than that in the mild subclinical hypothyroidism group. After 3 months of levothyroxine replacement therapy, serum TSH and TC levels of patients in both mild subclinical hypothyroidism and severe subclinical hypothyroidism groups were significantly decreased, which had no significant difference with the control group; Moreover, Person correlation analysis showed that TSH levels in subclinical hypothyroidism patients was significantly positively correlated with TC, These results suggested that the elevated TSH may be caused the abnormal TC of subclinical hypothyroidism patients.2. Compared with the control group, cPLA2 levels were significantly increased in mild and severe subclinical hypothyroidism group, and which were more apparent in the severe subclinical hypothyroidism group. Serum cPLA2 level of severe subclinical hypothyroidism patients significantly decreased after 3 months of levothyroxine replacement therapy; however, cPLA2 level of patients in severe subclinical hypothyroidism group was still higher than that in the normal control group. Results from correlation analysis showed that cPLA2 levels of subclinical hypothyroidism patients were positively correlated with TSH. Multiple regression analysis also showed that TSH and cPLA2 was most relevant. The relationship between TSH and cPLA2 was analyzed in vitro (peripheral mononuclear cells). The results showed that TSH could significantly increase the intracellular concentration and activity of cPLA2, and TSH and cPLA2 had a significant positive correlation. Relation between medium cPLA2 and intracellular cPLA2 was also analyzed. The results showed that medium cPLA2 level was significantly positively correlated with intracellular cPLA2 activity, which means that serum cPLA2 level may also reflect its activities in cells.3. Changes of many types of eicosanoids were detected in different subclinical hypothyroidism patients before and after replacement therapy.The results showed that major eicosanoids (11-dehydroTXB2,12-HETE and 8-isoPGF2a) levels of patients in mild or severe subclinical hypothyroidism group were significantly higher than that in the normal control group, the ratio of 11-dehydro TXB2/6-Keto PGF1a was also increased significantly. These results suggested that eicosanoids associated pathways of subclinical hypothyroidism patients were significantly abnormal, which resulted in significantly enhanced inflammation and stress response. Serum eicosanoid 8-isoPGF2a and 11-dehydroTXB2 dehydro levels, and the ratio of TXB2/6-Keto PGF1a were all significantly decreased in mild or severe subclinical hypothyroidism patients, after 3 months of levothyroxine replacement therapy.4. Correlation analysis about eicosanoids levels, thyroid hormone and lipid were analyzed to reveal the possible factors associated with multiple types of eicosanoids abnormalities of subclinical hypothyroidism patients. The results showed that 8-isoPGF2a was significantly positively correlated to TSH, cPLA2, TC, TG and LDL-C. While 6-Keto PGFla showed a significant negative correlation with TSH and TC, the correlation was weak.5-HETE had a weak but significant positive correlation with TC and LDL-C. In addition, TSH was significantly positively correlated to 11-dehydro-TXB2 and the ratio of 11-dehydro-TXB2/6-Keto PGFla. However, HDL-C, FT3 and other factors failed to find significant correlations with eicosanoids and the ratio of 11-dehydro-TXB2/6-Keto PGFla. These results also suggested that factors affected eicosanoid of subclinical hypothyroidism patients were complex, and the mechanisms all need to be studied.Conclusion:1、Short-term levothyroxine replacement therapy could ameliorate blood lipids and thyroid dysfunction of subclinical hypothyroidism patients.2、TSH and serum cPLA2 levels of subclinical hypothyroidism patients were significantly increased, and levothyroxine replacement therapy could correct this abnormality.3、Serum major eicosanoids significantly increased in subclinical hypothyroidism patients, and levothyroxine replacement therapy could correct this abnormality. Short-term replacement therapy might contribute to prevention of atherosclerosis by reducing inflammation, oxidative stress in subclinical hypothyroidism patients.