| Background:Subclinical hypothyroidism (SCH), which is defined as elevated serum thyroid-stimulating hormone (TSH) and normal serum free thyroxine (FT4), is becoming a global health problem because of its increasing prevalence and potential deleterious effects. SCH can be classified as mild (serum TSH concentrations of 4.5-10 mU/L) or significant (TSH≥10 mU/L), according to the size of the increase in the serum TSH. More and more population-based studies have demonstrated that both the risk and mortality of coronary heart disease are increased in SCH patients. Although the changes in the lipid profiles of SCH patients are still controversial, series of studies have reported increased level of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and triglycerides and decreased level of HDL-C in SCH patients. However, lipid abnormalities in hypothyroid patients do not fully account for the accelerated atherosclerosis and cardiovascular diseases, and other pathogenic factors may be involved. The changed lipid pattern may result in altered LDL oxidizability. It is known that oxidized LDL (oxLDL) is involved in the pathogenesis of atherosclerosis. Diekman et al reported that in hypothyroid state LDL is already peroxidized in the circulation due to the lack of sufficient antioxidant capacity provided by T4. However there is little knowledge about the susceptibility of LDL to oxidation in SCH patients.The oxidation of LDL converts it into an atherogenic form that contributes to the development of the atherosclerotic lesion. One of the major pathways of LDL oxidation is via the lipoxygenase (LOX) pathway by seeding molecules that include hydroperoxyoctadecadienoic acid (HPODE) and hydroperoxyeicosatetraenoic acid (HPETE). These hydroperoxy derivatives are subsequently reduced to their hydroxyl derivatives hydroxy-octadecadienoic acids (HODEs) and hydroxy-eicosatetraenoic acids (HETEs). For LDL, the oxidation of linoleic acid (LA) and arachidonic acid (AA) lead to the formation of HODE and HETE, respectively. Notably, these HODEs and HETEs enhance atherogenicity and play important roles in the pathogenesis of atherosclerosis. Today, most studies have focused on lipid peroxides issued from the non-enzymatic peroxidation of arachidonic acid. Little data is available on circulation lipid peroxides issued from linoleic acid, although it is the main polyunsaturated fatty acid (PUFA) in LDL. Except for LDL, Rago et al using UPLC-MS/MS method quantify the plasma concentrations of a panel of 18 key eicosanoids, and found that 9-HODE,13-HODE,8-HETE and LTB4 exhibited consistently lower levels in CAD patients compared to normal controls, and can be potential negative biomarkers for severe heart diseases. Today no data (to our knowledge) are available about changes of oxidation products of LA and AA either in LDL or plasma in subclinical hypothyroidism.Objectives:Considering that the SCH may increase the risk of atherosclerosis and cardiovascular diseases, and lipid peroxidation products may play important roles during atherogenesis, in the present study we attempted:1. To explore the status of lipid peroxidation in SCH patients as measured by concentrations of HODEs and HETEs both in plasma and LDL2. To explored the relationships among TSH levels, lipid peroxidation, and carotid intima-media thickness (IMT), a biomarker of subclinical atherosclerosis.3. To compare the differences of HODE and HETE levels in plasma and LDL respectively, attempting to identify a better surrogate marker for IMT. Subjects:The study subjects came from our epidemiological survey from Ningyang County, Shandong Province, from January to February 2013(n=1331). The diagnosis of SCH was based on finding high TSH levels associated with normal FT4 levels. Depending on the size of the increase in serum TSH, subclinical hypothyroidism can be classified as mild (serum TSH concentrations of 4.5-10 mU/L) or significant (TSH ≥10 mU/L). Based on the exclusion criteria (diabetes mellitus, hypertension, obesity (body mass index, BMI≥28 kg/m2), ischemic heart disease, history of or current smoking, active infection, malignancy, pituitary and rheumatologic diseases, and usage of drugs that affect the oxidant state or lipid parameters), we first included 160 newly diagnosed SCH patients. Patients’ thyroid function was measured twice for 6 months interval to rule out laboratory error or transient increases. Only 129 patients met the criteria of having two consistent results and made a final diagnosis of SCH. Among these, we selected 20 SCH patients aged between 18 and 65 years using the random number table method:10 mild SCH patients and 10 age- and sex- matched significant SCH patients. The control group consisted of 10 age- and sex-matched healthy euthyroid subjects from the same district.Methods:1. A doctoral questionnaire was used to record the patient’s general information, including gender, age, height, weight, systolic blood pressure(SBP), diastolic blood pressure(DBP) and waist circumference. Blood samples were collected from all patients between 8:00 A.M. and 10:00 A.M. after a minimum of a 10-h fast. Chemiluminescent procedures were employed to determine thyroid function, including TSH, free triiodothyronine (FT3) and free tetraiodothyronine (FT4). Automatic biochemical analyzer was used to detect the serum levels of TC, TG, LDL-C, HDL-C and glucose. Plasma was separated via centrifugation at 1500 g for 10 min with blood collected on EDTA and immediately frozen at -80℃ under nitrogen after 10μM BHT was added. All patients underwent a carotid IMT examination using a color ultrasonic diagnostic apparatus.2. A AKTA purifier system fast protein liquid chromatography (FPLC) equipped with a Superose 6 10/300GL column (GE Healthcare, Pittsburgh, PA, USA) was used to isolate LDL from plasma. The LDL peak was identified by determining the concentrations of TC and TG in each fraction using commercial kits. Also the basal parameters of isolated LDL were measured.3. The concentrations of HODEs and HETEs in both LDL and plasma were examined using liquid chromatograph-electrospray ionization-mass spectrometer (LC-ESI-MS) method.Statistical analysis:Statistical analyses were performed with SPSS version 17.0 for windows. One-way ANOVA with Bonferroni correction as the post-hoc test was used to seek differences in variables among groups. Significance was defined as a value of p< 0.05. If unstated, all values are expressed as the mean±SD for continuous variables. Correlation coefficients were determined using the Spearman correlation test.Results:1. Characteristics of the study groupsSerum LDL-C levels and mean-IMT in the significant SCH group were higher than they were in the euthyroid group (p<0.05), whereas no differences were found between the mild SCH and euthyroid groups (p>0.05). The TC content in LDL increased gradually but not significantly among the euthyroid, mild SCH, and significant SCH groups (p>0.05). The TG content in LDL was also not significantly different.2. A positive relationship between TSH and mean-IMT levelsAmong all subjects, linear and significant positive correlations were identified between TSH and mean-IMT (r=0.401,p=0.028). The correlations remained significant even after we adjusted for age, gender, BMI, TC, HDL-C, LDL-C, and TG (r=0.480, p=0.018). This finding suggests an association between SCH and the risk for atherosclerosis.3. Lipid peroxidation increased in SCH patientsThe concentrations of HODEs (both 9-HODE and 13-HODE) in plasma clearly increased in the significant SCH patients compared with the euthyroid subjects, and there was no difference between the mild SCH and euthyroid groups. Similar findings were also obtained with respect to the HODE concentrations in LDL. Compared with the euthyroid subjects, plasma 5-HETE and 12-HETE levels in the significant SCH patients were higher. No significant differences were detected with regard to the HETE concentrations in LDL among the 3 groups.4. HODEs in LDL were positively associated with TSHSpearman’s correlation analysis revealed that both 9-HODE (r=0.376,p=0.041) and 13-HODE (r=0.447,p=0.013) in LDL were linearly and positively correlated with TSH, which indicated that lipid peroxidation in LDL was significantly aggravated with the elevation of TSH.5. HODEs in LDL might be more sensitively indicative of atherosclerosisAll subjects were divided into two groups according to their IMT:normal (max-IMT≤0.9 mm) (n=16) and thickened (max-IMT> 0.9 mm) (w=14). The HODE concentrations (both 9-HODE and 13-HODE) in LDL were much higher in the thickened IMT group than they were in the normal group (p= 0.017 and 0.015, respectively). No difference was found in plasma HODE levels between the normal and thickened IMT groups. Moreover, there were no significant differences in the HETE concentrations (5-,12-, and 15-HETE) in either plasma or LDL between the normal and thickened IMT groups.In addition, a positive and significant correlation was obtained between mean-IMT and the concentration of 9-HODE in LDL (r=0.479, p=0.007); as well as the concentration of 13-HODE in LDL (r=0.447,p=0.013).Conclusions:1. With the elevation of TSH levels, SCH patients displayed lipid abnormality, aggravated lipid peroxidation and atherosclerosis, which suggest the close relations among SCH, lipid peroxidation and atherosclerosis.2. HODEs in LDL might be more sensitively indicative of atherosclerosis.3. Our results supported to treat patients with SCH and TSH levels≥10 mU/L to alleviate lipid peroxidation and development of atherosclerosis.Background:Subclinical hypothyroidism (SCH), which is defined as elevated serum thyroid-stimulating hormone (TSH) and normal serum free thyroxine (FT4), is becoming a global health problem because of its increasing prevalence and potential deleterious effects. More and more population-based studies have demonstrated that both the risk and mortality of coronary heart disease are increased in SCH patients. Although the changes in the lipid profiles of SCH patients are still controversial, series of studies have reported increased level of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and triglycerides and decreased level of HDL-C in SCH patients. However, lipid abnormalities in hypothyroid patients do not fully account for the accelerated atherosclerosis and cardiovascular diseases, and other pathogenic factors may be involved. The changed lipid pattern may result in altered LDL oxidizability. It is known that oxidized LDL (oxLDL) is involved in the pathogenesis of atherosclerosis. Diekman et al reported that in hypothyroid state LDL is already peroxidized in the circulation due to the lack of sufficient antioxidant capacity provided by T4. However there is little knowledge about the susceptibility of LDL to oxidation in SCH patients.The oxidation of LDL converts it into an atherogenic form that contributes to the development of the atherosclerotic lesion. One of the major pathways of LDL oxidation is via the lipoxygenase (LOX) pathway by seeding molecules that include hydroperoxyoctadecadienoic acid (HPODE) and hydroperoxyeicosatetraenoic acid (HPETE). These hydroperoxy derivatives are then reduced to their hydroxyl derivatives hydroxy-octadecadienoic acids (HODEs) and hydroxy-eicosatetraenoic acids (HETEs). For LDL, the oxidation of linoleic acid (LA) and arachidonic acid (AA) lead to the formation of HODE and HETE, respectively. Notably, these HODEs and HETEs enhance atherogenicity and play important roles in the pathogenesis of atherosclerosis. Today, most studies have focused on lipid peroxides issued from the non-enzymatic peroxidation of arachidonic acid. Little data is available on circulation lipid peroxides issued from linoleic acid, although it is the main polyunsaturated fatty acid (PUFA) in LDL. Except for LDL, Rago et al using UPLC-MS/MS method quantify the plasma levels of a panel of 18 key eicosanoids, and found that 9-HODE, 13-HODE,8-HETE and LTB4 exhibited consistently lower concentrations in CAD patients compared to normal controls, and can be potential negative biomarkers for severe heart diseases. Today no data (to our knowledge) are available about changes of oxidation products of LA and AA either in LDL or plasma in subclinical hypothyroidism.Objectives:Considering that the SCH may increase the risk of atherosclerosis and cardiovascular diseases, and lipid peroxidation products may play important roles during atherogenesis, in the present study we attempted:1. To explore the status of lipid peroxidation in SCH patients as measured by concentrations of HODEs and HETEs both in plasma and LDL.2. To explored the relationships among TSH levels, lipid peroxidation, and carotid intima-media thickness (IMT), a biomarker of subclinical atherosclerosis.3. To compare the differences of HODE and HETE levels in plasma and LDL respectively, attempting to identify a better surrogate marker for IMT.SubjectsThe study subjects came from our epidemiological survey from Ningyang County, Shandong Province, from January to February 2013(n=1331). The diagnosis of SCH was based on finding high TSH levels associated with normal FT4 levels. SCH can be classified as mild (serum TSH concentrations of 4.5-10 mU/L) or significant (TSH≥10 mU/L), according to the size of the increase in serum TSH. Based on the exclusion criteria (diabetes mellitus, hypertension, obesity (body mass index, BMI≥28 kg/m2), ischemic heart disease, history of or current smoking, active infection, malignancy, pituitary and rheumatologic diseases, and usage of drugs that affect the oxidant state or lipid parameters), we first included 160 newly diagnosed SCH patients. Patients’thyroid function was measured twice for 6 months interval to rule out laboratory error or transient increases. Only 129 patients met the criteria of having two consistent results and made a final diagnosis of SCH. Among these, we selected 20 SCH patients aged between 18 and 65 years using the random number table method:10 mild SCH patients and 10 age-and sex-matched significant SCH patients. The control group consisted of 10 age-and sex-matched healthy euthyroid subjects from the same district.Methods6. A doctoral questionnaire was used to record the patient’s general information, including gender, age, height, weight, systolic blood pressure(SBP), diastolic blood pressure(DBP) and waist circumference. After a minimum of a 10-h fast, blood samples were collected from all sujects between 8:00 A.M. and 10:00 A.M.. Thyroid function was determined using chemiluminescent procedures, including free triiodothyronine (FT3), free tetraiodothyronine (FT4) and TSH. Automatic biochemical analyzer was used to detect the serum levels of TC, TG, LDL-C,HDL-C and glucose. Plasma was separated via centrifugation at 1500 g for 10 min with blood collected on EDTA and immediately frozen at -80℃ under nitrogen after 10μM BHT was added. All patients underwent a carotid IMT examination using a color ultrasonic diagnostic apparatus.7. A AKTA purifier system fast protein liquid chromatography (FPLC) equipped with a Superose 610/300GL column (GE Healthcare, Pittsburgh, PA, USA) was used to isolate LDL from plasma. The LDL peak was identified by determining the concentrations of TC and TG in each fraction using commercial kits. Also the basal parameters of isolated LDL were measured.8. The concentrations of HODEs and HETEs in both LDL and plasma were examined using liquid chromatograph-electrospray ionization-mass spectrometer (LC-ESI-MS) method.Statistical analysisStatistical analyses were performed with SPSS version 17.0 for windows. One-way ANOVA with Bonferroni correction as the post-hoc test was used to seek differences in variables among groups. Significance was defined as a value of p 0.05. If unstated, all values are expressed as the mean±SD for continuous variables. Correlation coefficients were determined using the Spearman correlation test.Results1. Characteristics of the study groupsSerum LDL-C levels and mean-IMT in the significant SCH group were higher than they were in the euthyroid group (p<0.05), whereas no differences were found between the mild SCH and euthyroid groups (p>0.05). The TC content in LDL increased gradually but not significantly among the euthyroid, mild SCH, and significant SCH groups (p>0.05). The TG content in LDL was also not significantly different.2. A positive relationship between TSH and mean-IMT levelsAmong all subjects, linear and significant positive correlations were identified between TSH and mean-IMT (r=0.401, p=0.028). The correlations remained significant even after we adjusted for age, gender, BMI, TC, HDL-C, LDL-C, and TG (r=0.480, p=0.018). This finding suggests an association between SCH and the risk for atherosclerosis.3. Lipid peroxidation increased in SCH patientsThe concentrations of HODEs (both 9-HODE and 13-HODE) in plasma clearly increased in the significant SCH patients compared with the euthyroid subjects, and there was no difference between the mild SCH and euthyroid groups. Similar findings were also obtained with respect to the HODE concentrations in LDL. Compared with the euthyroid subjects, plasma 5-HETE and 12-HETE levels in the significant SCH patients were higher. No significant differences were detected with regard to the HETE concentrations in LDL among the 3 groups.9. HODEs in LDL were positively associated with TSHSpearman’s correlation analysis revealed that both 9-HODE (r=0.376, p=0.041) and 13-HODE (r=0.447, p=0.013) in LDL were linearly and positively correlated with TSH, which indicated that lipid peroxidation in LDL was significantly aggravated with the elevation of TSH.10. HODEs in LDL might be more sensitively indicative of atherosclerosisAll subjects were divided into two groups according to their IMT:normal (max-IMT≤0.9 mm) (n=16) and thickened (max-IMT> 0.9 mm) (w=14). The HODE concentrations (both 9-HODE and 13-HODE) in LDL were much higher in the thickened IMT group than they were in the normal group (p= 0.017 and 0.015, respectively). No difference was found in plasma HODE levels between the normal and thickened IMT groups. Moreover, there were no significant differences in the HETE concentrations (5-,12-, and 15-HETE) in either plasma or LDL between the normal and thickened IMT groups.In addition, a positive and significant correlation was obtained between mean-IMT and the concentration of 9-HODE in LDL (r=0.479, p=0.007); as well as the concentration of 13-HODE in LDL (r=0.447, p=0.013).Conclusions4. With the elevation of TSH levels, SCH patients displayed lipid abnormality, aggravated lipid peroxidation and atherosclerosis, which suggested the close relations among SCH, lipid peroxidation and atherosclerosis.5. HODEs in LDL might be more sensitively indicative of atherosclerosis.6. Our results supported to treat patients with SCH and TSH levels≥10 mU/L to alleviate lipid peroxidation and development of atherosclerosis. |
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