Serum Homocysteine in Thyroiditis Treated With Levothyroxine
Serum Homocysteine in Thyroiditis Treated With Levothyroxine
Thirty one euthyroid women treated with levothyroxine (L-T4) due to Hashimoto thyroiditis (HT) at the outpatient clinic of the Department of Endocrinology, Metabolism and Internal Medicine and twenty six females with chronic autoimmune thyroiditis in euthyroidism without L-T4 replacement therapy were enrolled in the study (Table 1). All women with HT had positive TPOAbs. Forty healthy females negative for TPOAbs comparable for age and body mass index (BMI) participated in the study as controls (Table 1).
All subjects and controls were euthyroid, either spontaneously, or under L-T4 medication. None of the patients and the controls had a history of any acute or chronic disease, including diabetes mellitus, hypertension, angina pectoris, evidence of any kidney or liver disorder. Exclusion criteria were also use of any medications (including oral contraceptives and vitamin supplements), smoking, alcoholism.
All subjects underwent physical examination with recording of height, weight, heart rate, systolic and diastolic blood pressure. Blood samples were obtained after an overnight fast. In patients with HT blood samples were drawn before the ingestion of usual morning L-T4 medication. Serum levels of thyrotropin hormone (TSH), free thyroxine (FT4), TPOAbs, Hcy, total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), triacylglycerol (TAG) were evaluated.
TSH and FT4 were measured using the electrochemiluminescence technique in Cobas E 601 (norm ranges: TSH 0.27–4.2 mU/l; FT4 11.5–21.0 pmol/l). TPOAbs were measured by radioimmunoassay (norm range: <34 IU/ml).
Serum Hcy levels were assessed by high-performance liquid chromatography (HPLC). The analyzed plasma thiol compounds (Hcy, Fluka Germany) were diluted with water at 2:1 ratio and reduced using 1% TCEP (Tris-(2-carboxyethyl)-phosphin-hydrochloride; Applichem, Germany) at 1:9 ratio. Subsequently, the sample was deproteinized using 1 M HClO4 (at 2:1 ratio) and applied to the HPLC/EC system.
To determine thiol concentration the samples were fed to the HPLC system (P580A; Dionex, Germany) coupled to an electrochemical detector (CoulArray 5600; ESA, USA). The analysis was performed in Termo Hypersil BDS C18 column (250 mm × 4.6 mm × 5 μm) (Germany) in isocratic conditions, using the mobile phase of 0.15 M phosphate buffer, pH 2.9, supplemented with 12.5–17% acetonitrile. The system was controlled, and the data were collected and processed using Chromeleon software (Dionex, Germany).
The study was approved by the Poznan University of Medical Sciences Ethical Commitee, and informed consent was signed by every subject.
Comparison of analyzed parameters between three groups (HT patients versus healthy controls) was performed by Kruskal-Wallis test with Dunn's post-hoc tests because data did not follow normal distribution. Normality was analyzed by Shapiro-Wilk's test. Results were presented as medians and interquartile ranges (IQR). Spearman's correlation coefficient was used to measure the strength of relationship of analyzed parameters. All tests were performed two-tailed and were considered as significant at p < 0.05. Statistical analyses were performed with Statistica 10 (StatSoft Inc., Poland) and MedCalc version 10.3.2 (MedCalc Software, Mariakerke, Belgium) .
Methods
Thirty one euthyroid women treated with levothyroxine (L-T4) due to Hashimoto thyroiditis (HT) at the outpatient clinic of the Department of Endocrinology, Metabolism and Internal Medicine and twenty six females with chronic autoimmune thyroiditis in euthyroidism without L-T4 replacement therapy were enrolled in the study (Table 1). All women with HT had positive TPOAbs. Forty healthy females negative for TPOAbs comparable for age and body mass index (BMI) participated in the study as controls (Table 1).
All subjects and controls were euthyroid, either spontaneously, or under L-T4 medication. None of the patients and the controls had a history of any acute or chronic disease, including diabetes mellitus, hypertension, angina pectoris, evidence of any kidney or liver disorder. Exclusion criteria were also use of any medications (including oral contraceptives and vitamin supplements), smoking, alcoholism.
All subjects underwent physical examination with recording of height, weight, heart rate, systolic and diastolic blood pressure. Blood samples were obtained after an overnight fast. In patients with HT blood samples were drawn before the ingestion of usual morning L-T4 medication. Serum levels of thyrotropin hormone (TSH), free thyroxine (FT4), TPOAbs, Hcy, total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), triacylglycerol (TAG) were evaluated.
TSH and FT4 were measured using the electrochemiluminescence technique in Cobas E 601 (norm ranges: TSH 0.27–4.2 mU/l; FT4 11.5–21.0 pmol/l). TPOAbs were measured by radioimmunoassay (norm range: <34 IU/ml).
Serum Hcy levels were assessed by high-performance liquid chromatography (HPLC). The analyzed plasma thiol compounds (Hcy, Fluka Germany) were diluted with water at 2:1 ratio and reduced using 1% TCEP (Tris-(2-carboxyethyl)-phosphin-hydrochloride; Applichem, Germany) at 1:9 ratio. Subsequently, the sample was deproteinized using 1 M HClO4 (at 2:1 ratio) and applied to the HPLC/EC system.
To determine thiol concentration the samples were fed to the HPLC system (P580A; Dionex, Germany) coupled to an electrochemical detector (CoulArray 5600; ESA, USA). The analysis was performed in Termo Hypersil BDS C18 column (250 mm × 4.6 mm × 5 μm) (Germany) in isocratic conditions, using the mobile phase of 0.15 M phosphate buffer, pH 2.9, supplemented with 12.5–17% acetonitrile. The system was controlled, and the data were collected and processed using Chromeleon software (Dionex, Germany).
The study was approved by the Poznan University of Medical Sciences Ethical Commitee, and informed consent was signed by every subject.
Comparison of analyzed parameters between three groups (HT patients versus healthy controls) was performed by Kruskal-Wallis test with Dunn's post-hoc tests because data did not follow normal distribution. Normality was analyzed by Shapiro-Wilk's test. Results were presented as medians and interquartile ranges (IQR). Spearman's correlation coefficient was used to measure the strength of relationship of analyzed parameters. All tests were performed two-tailed and were considered as significant at p < 0.05. Statistical analyses were performed with Statistica 10 (StatSoft Inc., Poland) and MedCalc version 10.3.2 (MedCalc Software, Mariakerke, Belgium) .