Changes in metabolic and cardiovascular risk factors before and after treatment in overt hypothyroidism

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1 ORIGINAL ARTICLE Changes in metabolic and cardiovascular risk factors before and after treatment in overt hypothyroidism Faruk Kutluturk 1, Suleyman Yuce 2, Turker Tasliyurt 2, Berna Murat Yelken 3, Pelin Aytan 2, Banu Ozturk 2, Abdulkerim Yılmaz 2 1 Department of Endocrinology and Metabolism, 2 Department of Internal Medicine, 3 Department of Nephrology; Gaziosmanpasa University, School of Medicine, Tokat, Turkey. ABSTRACT Aim Overt hypothyroidism is associated with an increased risk for developing cardiovascular disease. We aimed to assess the changes in renal function, serum lipids, vitamin B12, folic acid and homocysteine levels before and after treatment in hypothyroid patients. Corresponding author: Faruk Kutluturk Department of Endocrinology and Metabolism Gaziosmanpasa University, School of Medicine Kaleardi, Muhuttin Fusinoglu St Tokat, Turkey Phone: ; Fax: ) ; fkutluturk@yahoo.com Original submission: 12 December 2012; Revised submission: 06 February 2013; Accepted: 15 February Methods The study included 54 patients (F/M=47/7) with overt hypothyroidism. All patients were assessed for demographic characteristics such as age, gender, body weight, and body mass index. Fasting blood samples were taken from the patients for analysis of chemical parameters including thyroid stimulating hormone (TSH), free thyroxine (ft4), homocysteine, total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), folic acid, and vitamin B12 levels before and after L-thyroxine (LT4) treatment. Results Homocysteine levels in hypothyroidism (9.67±5.24 mmol/l) were significantly higher than in euthyroid state (8.16±3.38 mmol/l, p=0.038). Glomerular filtration rate (GFR) was lower before treatment. Following LT4 replacement, renal functions significantly improved. After achieving the euthyroid state, folic acid levels significantly increased although vitamin B12 levels were not changed. There was a significant reduction in serum lipid levels after LT4 replacement. It was demonstrated that there was a significant negative correlation between GFR and lipids and a positive correlation with homocysteine and lipids at hypothyroid state. After normalization of thyroid functions, the correlations became non-significant. Conclusion The hypothyroidism was associated with increased serum homocysteine, lipids, and creatinine concentrations. The improvement of these parameters with LT4 replacement may be associated with the lower risk for atherosclerotic cardiovascular diseases in the patients with hypothyroidism. Key words: thyroid disease, homocysteine, lipids, atherosclerosis Med Glas (Zenica) 2013; 10(2):

2 Kutluturk et al. Thyroid function and cardiometabolic risk factors INTRODUCTION Hypothyroidism is associated with an increase in a number of atherosclerotic coronary artery disease risk factors including dyslipidemia, hypertension, and elevated levels of homocysteine (1-3). Hyperhomocysteinemia, as an independent risk factor for cardiovascular disease, is thought to be responsible for about 10 percent of the total risk (4-8). Based on various calculation models, reduction of elevated plasma homocysteine concentrations may prevent up to 25 percent of cardiovascular events (4). Hyperhomocysteinemia may accelerate atherosclerosis by increasing the oxidation of low-density lipoprotein and by endothelial dysfunction (1, 5, 7, 9). The recent clinical trials have shown that plasma total homocysteine levels were elevated in patients with overt hypothyroidism (10, 11). Homocysteine is a sulphur-containing amino acid biosynthesized during the conversion of methionine to cysteine. Folic acid and vitamin B12 are required for the remethylation of homocysteine to methionine; vitamin B6 is required for the transsulfuration of homocysteine to cysteine (12). Folic acid, vitamin B12 and vitamin B6 deficiencies and reduced enzyme activities inhibit the breakdown of homocysteine, thus increase the concentration of intracellular homocysteine (13). Experimental studies indicated that methylenetetrahydrofolate reductase is decreased in hypothyroidism (10-12). Furthermore, elevated serum homocysteine levels are thought to be due to impaired renal metabolism or reduced urinary excretion of homocysteine in hypothyroid patients. The haemodynamic effects of hypothyroidism are probable reasons of reduced renal blood flow and glomerular filtration rate (14). The aim of our study was to assess the changes in renal function, serum lipids, vitamin B12, folic acid and homocysteine levels before and after the treatment of patients with overt hypothyroidism. PATIENTS AND METHODS This study included 54 patients (female/ male=47/7, age range years) with overt hypothyroidism (TSH levels >10 μiu/l, and free thyroxine (ft4) levels <0.93 ng/dl). Normal ranges in our laboratory are as follows: TSH, μiu/ l, ft4, ng/dl. Hypothyroidism was due to thyroid surgery in 23 (43%) and Hashimoto thyroiditis in 31 (57%) patients. Exclusion criteria were: use of any drug that interferes with homocysteine metabolism (such as folic acid, vitamin B12, and B6 antagonists, oral contraception, anticonvulsants, thiazides, fibrate, antifolates, anticonvulsant agents, tamoxifen, and theophylline), use of statins and anti-thyroid medications, history of diabetes mellitus, pregnancy, cancer, anemia, renal disease (such as nephrotic syndrome, end stage renal failure), liver disease, familial hypercholesterolemia, chronic alcoholism and systemic illness (such as malabsorbtion syndromes, inflammatory bowel diseases). All patients were assessed for demographic characteristics such as age, gender, body weight and body mass index. Informed consent was obtained from all patients. The study protocol was approved by the Regional Ethics Committee. Serum samples were taken from the patients for analysis of chemical parameters including homocysteine, total cholesterol, triglyceride (TG), high-density lipoprotein cholesterol (HDL), fasting blood glucose, folic acid and vitamin B12 levels in hypothyroid state before the treatment and euthyroid state after LT4 treatment. Normal ranges in our laboratory are: folic acid, 3-17 ng/ ml, vitamin B12, pg/ml, homocysteine, 0-15 µmol/l. An initial dose of mcg/d of LT4 was used and dose titration was adjusted according to the TSH levels every 6-8 weeks. Second blood analysis was done in euthyroid state, after 2 3 months of the treatment with LT4 in individualized doses. A fasting blood sample collected into an EDTA tube was used for the measurement of plasma homocysteine. Serum homocysteine concentration was measured by high-performance liquid chromatography. Vitamin B12 and folic acid levels were measured by electrochemiluminescence immunoassay. The hormonal assessment included the measurement of serum TSH, and ft4, and was performed using Cobas e601 chemiluminescent analyzer (Roche Diagnostics). Serum total cholesterol, HDL and TG were determined enzymatically (Olympus Diagnostica, Lismeehan, Ireland). LDL was calculated with the Friedewald s formula (15): LDL=total cholesterol-(hdl+tg/5). Serum creatinine was measured by an automated enzymatic method (Olym- 349

3 Medicinski Glasnik, Volume 10, Number 2, August 2013 pus Diagnostica, Ireland). Estimated glomerular filtration rate (GFR) was calculated using the 4v-MDRD formula (170 x Scr x age x (0.762 if female) x (1.180 if black) xsu x Alb+0.318) in both hypothyroid and euthyroid state. Normal ranges of GFR are ml/min. The relation of homocysteine levels with age, body mass index, serum lipids, fasting blood glucose, plasma folic acid, and vitamin B12 levels were also evaluated. Quantitative variables distributions were analyzed using Kolmogorov-Smirnov test. Parametric tests were applied when test results were normally distributed. When the quantitative variables had distribution different from normal, non-parametric tests were used. Parametric variables were described as means±standard deviations (SD). Nonparametric variables were presented as means, minimum-maximum. Qualitative variables were described as counts and proportions. For parametric variables, ANOVA was applied to evaluate the differences among groups. Comparison between groups of non-parametric variables was performed by using Kruskal-Wallis test. Multivariate analysis was performed in order to weight the burden of confounding factors on results (TSH, BMI, vitamin B12, folic acid, lipids). A p value <0.05 was considered as statistically significant. RESULTS A total of 54 (male/female=7/47) patients were enrolled in this study. Mean age was 46.5±11.9 (range 20-75) years. Serum homocysteine, lipid levels and renal functions were evaluated in patients with hypothyroidism. Second blood analysis was done in euthyroid state, after 3 6 months of treatment with LT4 in individualized doses (Table 1). Mean homocysteine levels in patients with hypothyroidism before the treatment (9.67±5.24 µmol/l) were significantly higher than in euthyroid state (8.16±3.38 µmol/l, p=0.038). The mean levels of serum creatinine were higher and calculated GFR values were lower before the treatment. Following LT4 replacement, serum creatinine decreased with an increase in GFR. GFR was increased for about 13% with LT4 replacement (Figure 1). After recovery of thyroid functions with LT4 replacement, serum folic acid levels significantly increased (p=0.017) although vitamin B12 levels were not changed. Table 1. Clinical and laboratory characteristics of patients in hypothyroid and euthyroid state. Characteristic Hypothyroid state Euthyroid state BMI (kg/m2) ± ± * Body Weight (kg) ± ± * TSH (μiu/ l) ± ± 1.55 <0.001* ft4 (ng/dl) 0.64 ± ± 0.29 <0.001* Creatinine (mg/dl) 0.78 ± ± 0.13 <0.001* Homocysteine (µmol/l) 9.67 ± ± * BUN (mg/dl) ± ± GFR (ml/min/1.73m 2 ) ± ± <0.001* Total-C (mg/dl) ± ± <0.001* LDL- C (mg/dl) ± ± <0.001* HDL- C (mg/dl) ± ± * Triglyceride (mg/dl) ± ± Vitamin B12 (pmol/l) ± ± Folic acid (ng/ml) 7.61 ± ± * *p<0.05; BMI, body mass index; TSH, thyroid stimulating hormone; ft4, free thyroxine; GFR, glomerular filtration rate; BUN, blood urea nitrogen; LDL, low-density lipoprotein cholesterol; HDL, high-density lipoprotein cholesterol Figure 1. The GFR values of patient group in euthyroid and hypothyroid state. Mean GFR was increased for about 13% with LT4 replacement (p<0.001) The correlations between plasma homocysteine/ GFR and other parameters in hypothyroid patients before the treatment were summarized at Table 2. A negative correlation between GFR and age, serum TSH and ft4 levels was found. No correlation was found between GFR and BMI, vitamin B12, and folic acid levels. Furthermore there was no significant correlation between homocysteine and BMI, folic acid and vitamin B12 levels. There was a correlation between homocysteine and GFR, but not statistically significant (p=0.07). However, a significant positive correlation between homocysteine and both TSH and creatinine was demonstrated. The mean serum levels of total cholesterol, LDL and HDL cholesterol were significantly higher in the state of hypothyroidism than euthyroidism. After the treatment with LT4 serum total cholesterol, LDL and HDL cholesterol levels statistically significant decreased without statin treatment (Table 1). There was a positive correlation p 350

4 Kutluturk et al. Thyroid function and cardiometabolic risk factors Table 2. Correlations between serum homocysteine levels, GFR and other parameters in hypothyroid patients before treatment. Characteristic Homocysteine GFR (MDRD) r p r p Age <0.001* Body Weight BMI TSH <0.001* <0.001* ft * * BUN <0.001* Creatinine * <0.001* GFR Homocysteine Vitamin B Folic acid Total-C * * LDL-C * * HDL-C Triglyceride * r: pearson correlation. BMI, body mass index; TSH, thyroid stimulating hormone; ft4, free thyroxine; GFR, glomerular filtration rate; BUN, blood urea nitrogen; LDL, low-density lipoprotein cholesterol; HDL, high-density lipoprotein cholesterol; *p<0.05 between homocysteine and total cholesterol and LDL cholesterol (p=0.027, p=0.048, respectively; Table 2). Moreover, a strong negative association between GFR and total cholesterol, LDL and TG levels was indicated (p=0.005, p=0.004, and p=0.017, respectively; Table 2). After the LT4 replacement, we did not find any correlation between serum lipids levels and homocysteine levels, GFR values in euthyroid state. A positive correlation with homocysteine levels and total cholesterol, LDL in patients with overt hypothyroidism was demonstrated. This correlation became non-significant after LT4 replacement. A multiple linear regression model was used to identify independent predictor of homocysteine levels and GFR. The linear regression model with stepwise selection process showed that only TSH levels were significantly and independently associated with homocysteine (p=0.005) and GFR (p<0.001) in the patients with hypothyroidism. DISCUSSION The present study showed that patients with overt hypothyroidism had had higher serum homocysteine, creatinine and lipid levels than euthyroid state. Serum homocysteine levels of the patients with overt hypothyroidism significantly decreased with L-thyroxine treatment. This study revealed positive relationship between hypothyroidism and both hypercholesterolemia and hyperhomocysteinemia. Elevated homocysteine levels may have a multiplicative effect on cardiovascular risk factors in patients with hyperlipidemia and hypertension (2, 16, 17). Evidence from epidemiological studies suggested an increased risk for venous thrombosis with elevated homocysteine concentrations (4, 9, 18). Due to atherosclerotic lipid profile, the patients with hypothyroidism have suffered from cardiovascular morbidity. Moreover, McQuade et al. (3) showed that not only overt hypothyroidism but also moderate subclinical hypothyroidism are associated with increased risk of coronary heart disease and it was suggested that the patients with moderate subclinical patients should be treated with thyroid replacement therapy. Several studies indicated that overt hypothyroidism was associated with increased serum homocysteine concentrations and also, with LT4 replacement, homocysteine levels were normalized (19, 20). Our study confirmed the previous data, the patients with overt hypothyroidism had higher homocysteine levels and after normalization of thyroid functions, homocysteine levels decreased significantly. In the largest published study on plasma homocysteine and hyperthyroidism, normalization of thyroid status was associated with a significant increase of homocysteine; in correlation analysis, a significant correlation with ft4 was observed (21). Experimental studies have shown that the activity of flavoprotein methylene tetrahydrofolate reductase (MTHFR), the enzyme that participates in folic acid metabolism, is influenced by thyroid status (11, 22). Hepatic activity of MTHFR is increased in hyperthyroid state and decreased in hypothyroid state. This mechanism could explain the changes in plasma homocysteine levels in the patients with thyroid dysfunction and alternations after treatment. Thyroid hormones influence renal blood flow, GFR, and the function of many transport systems along the nephron, and sodium and water homeostasis (6, 23). These effects are related to the severity of thyroid dysfunction. Our study demonstrated that hypothyroidism was associated with low GFR and after the normalization of thyroid functions GFR increased for 13% percent. Moreover, serum TSH levels were found as significant and independent variable associated with GFR in the present study. Kidney is the major site for the removal and metabolism of homocysteine (24). Even a mild 351

5 Medicinski Glasnik, Volume 10, Number 2, August 2013 reduction in GFR leads to increased levels of homocysteine (25). A positive correlation between homocysteine and serum creatinine concentrations was reported previously. In our study, serum creatinine and homocysteine levels were elevated in overt hypothyroidism and after achieving euthyroid state with LT4 treatment, both of these levels were decreased significantly. Also we found a strong correlation with homocysteine and creatinine levels. It was demonstrated that serum total cholesterol and LDL levels were significantly decreased without statin treatment after the treatment with LT4 (26). As shown by the previous reports, high TSH is associated with deleterious changes in serum lipids as risk factors for atherosclerosis and coronary heart disease (27). Thyroid hormones are well known to have effects on the transport of the plasma lipoproteins and stimulate LDL receptor activity and in this way, cholesterol and LDL typically accumulate in plasma of patients with hypothyroidism (27). Danese et al. (28) reviewed the results of the total of 13 studies about LT4 effect on lipids in mild thyroid failure. In this review decreased serum total cholesterol concentration was reported in 11 of 13 studies, whereas decreased serum LDL cholesterol concentration was reported in 7 of 9 studies. Our results suggest that there was a positive correlation between serum lipids (total cholesterol and LDL) and homocysteine. Moreover, we showed a strong negative association between GFR and total cholesterol, LDL and TG. After normalization of thyroid functions with LT4 replacement the correlation between serum lipids and GFR had lost the significance. According to our knowledge there was limited data about this subject in the literature. REFERENCES 1. Veeranna V, Zalawadiya SK, Niraj A, Pradhan J, Ference B, Burack RC, Jacop S, Afonso L. Homocysteine and reclassification of cardiovascular disease risk. J Am Coll Cardiol 2011; 58: Duntas LH, Brenta G. The effect of thyroid disorders on lipid levels and metabolism. Med Clin North Am 2012; 96: McQuade C, Skugor M, Brennan DM, Hoar B, Stevenson C, Hoogwerf BJ. Hypothyroidism and moderate subclinical hypothyroidism are associated with increased all-cause mortality-a precis database study. Thyroid 2011; 21: In this study there was no relationship between homocysteine and vitamin B12 levels. Vitamin deficiency leads to reduced remethylation and elevated serum homocysteine. In hypothyroid patients, there were different reports, some studies demonstrated reduced (6, 29) while others reported unchanged levels (24) of vitamin B12. Some guidelines recommend supplementation of folic acid and B group vitamins or water soluble vitamins in people with kidney disease (30, 31). However, in the large systematic review by Jardine et al. (32) folic acid based homocysteine lowering treatment did not prevent cardiovascular events and mortality, requirement for dialysis treatment, or access thrombosis. It has been reported that hypothyroid patients have lower folic acid levels (6, 24), and concluded that homocysteine in hypothyroidism is associated with altered folic acid status. Similarly, in the hypothyroid state, the folic acid levels were lower than in the euthyroid state and increased after treatment in our study. In conclusion, the hypothyroid state was associated with increased serum homocysteine, lipids, and creatinine concentrations. The improvement of these parameters with LT4 replacement may be associated with the lower risk for atherosclerotic cardiovascular diseases in patients with hypothyroidism. Further studies will be necessary to clarify the atherosclerosis-hypothyroidism relation. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATIONS Competing interests: none to declare Stanger O, Herrmann W, Pietrzik K, Fowler B, Geisel J, Dierkes J, Weber M. Clinical use and rational management of homocysteine, folic acid and B vitamins in cardiovascular and thrombotic diseases. Z kardiol 2004; 93: Boushey CJ, Beresford SA, Omenn GS, Motulsky AG. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. Probable benefits of increasing folic acid intakes. J Am Med Assoc 1995; 274: Orzechowska-Pawilojc A, Siekierska-Hellmann M, Syrenicz A, Sworczak K. Homocysteine, folate, and cobalamin levels in hyperthyroid women before and after treatment. Endokrynol Pol 2009; 60:

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