Assessment of serum apelin and lipocalin-2 levels in patients with subclinical hypothyroidism

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ScienceDirect www.sciencedirect.com Annales d’Endocrinologie 75 (2014) 10–14

Original article

Assessment of serum apelin and lipocalin-2 levels in patients with subclinical hypothyroidism Évaluation des niveaux d’apéline et de lipocalin-2 sériques chez les patients atteints d’hypothyroïdie infraclinique Mehmet Zorlu a,∗ , Muharrem Kiskac a , Cumali Karatoprak a , Sidika Kesgin b , Mustafa Cakirca a , Kemal Yildiz a , Cuneyt Ardic c , Mehmet Ali Cikrikcioglu a , Reha Erkoc a b

a Faculty of medicine, Bezmialem Vakif university, internal medicine clinic, 34093 Fatih, Istanbul, Turkey Department of medical biochemistry clinic, faculty of medicine, Bezmialem Vakif University, 34093 Fatih, Istanbul, Turkey c Family health care center, 53100 Rize, Turkey

Abstract Objectives. – Subclinical hypothyroidism is the precursor to hypothyroidism because it has a tendency to transform into hypothyroidism. Subclinical hypothyroidism is considered one of the risk factors causing metabolic syndrome. Metabolic syndrome can be characterized by plasma levels of apelin and lipocalin-2, both released from adipocytes. In the present study, we aimed to measure serum apelin and lipocalin-2 levels of patients with subclinical hypothyroidism and compare them with serum apelin and lipocalin-2 levels from healthy individuals. Methods. – This was a cross-sectional study. A total of 80 subjects were enrolled in the study and divided into two groups: Group A included 39 patients (females, n = 34) diagnosed with subclinical hypothyroidism, and Group B (the control group) comprised 41 healthy volunteers (females, n = 38). Serum samples were obtained from each participant for the measurement of apelin and lipocalin-2. These were then stored at minus 80 ◦ C until the time of analysis, when serum apelin and lipocalin-2 levels of the two groups were compared. Results. – Patients with subclinical hypothyroidism (Group A and Group B subjects [healthy controls]) were comparable with respect to gender, age, and body mass index (BMI) (P = 0.412, P = 0.863, and P = 0.269, respectively), nor was there a statistically significant difference between groups in terms of apelin and lipocalin-2 levels (P = 0.87, and P = 0.67, respectively). Apelin levels showed a positive and significant correlation with BMI (P = 0.034). Serum lipocalin-2 levels showed significant positive correlations with BMI and creatinine levels (P = 0.002, and P = 0.025, respectively). Conclusion. – In the present study, no significant difference of serum apelin and lipocalin-2 levels was observed between patients with subclinical hypothyroidism and healthy control subjects. Positive correlations were found, however, between serum apelin level and BMI as well as between serum lipocalin-2 and BMI and creatinine levels. © 2014 Published by Elsevier Masson SAS. Résumé Objectifs. – L’hypothyroïdie infraclinique est le précurseur de l’hypothyroïdie, dans la mesure où elle a tendance à se transformer en hypothyroïdie. Elle est considérée comme l’un des facteurs de risque provoquant le syndrome métabolique. Celui-ci se caractérise par des taux plasmatiques d’apéline et de lipocaline-2, tous deux libérés par les adipocytes. Dans la présente étude, nous avons cherché à mesurer les niveaux d’apéline et de lipocaline-2 sériques chez des patients atteints d’hypothyroïdie infraclinique et à les comparer avec ceux d’individus sains. Méthodes. – Il s’agit d’une étude transversale. Quatre-vingt sujets ont été inclus dans l’étude et divisés en deux groupes : le groupe A comprenait 39 patients (femmes, n = 34) atteints d’hypothyroïdie infraclinique, et le groupe B (groupe témoin) 41 volontaires sains (femmes, n = 38). Des échantillons de sérum ont été obtenus à partir de chaque participant pour la mesure de l’apéline et de la lipocaline-2. Ceux-ci ont ensuite été stockés à –80◦ C jusqu’au moment de l’analyse des niveaux d’apéline et de lipocaline-2 sériques des deux groupes ont été comparés. Résultats. – Les patients atteints d’hypothyroïdie infraclinique (groupe A) et les sujets du groupe B (témoins sains) sont comparables en ce qui concerne le sexe, l’âge

∗

Corresponding author. E-mail addresses: [email protected], [email protected] (M. Zorlu), dr [email protected] (M. Kiskac), [email protected] (C. Karatoprak), [email protected] (S. Kesgin), [email protected] (M. Cakirca), [email protected] (K. Yildiz), [email protected] (C. Ardic), [email protected] (M.A. Cikrikcioglu), [email protected] (R. Erkoc). 0003-4266/$ – see front matter © 2014 Published by Elsevier Masson SAS. http://dx.doi.org/10.1016/j.ando.2013.12.001

M. Zorlu et al. / Annales d’Endocrinologie 75 (2014) 10–14

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et l’indice de masse corporelle (IMC) (p = 0,412, p = 0,863 et p = 0,269, respectivement) ; de même, il n’existe pas de différence statistiquement significative entre les groupes s’agissant des niveaux d’apéline et de lipocaline (p = 0,87 et p = 0,67, respectivement). Les niveaux d’apéline ont montré une corrélation positive et significative avec l’IMC (p = 0,034). Les niveaux de lipocaline-2 sérique ont montré des corrélations positives significatives avec les taux de créatinine et l’IMC (p = 0,002 et p = 0,025, respectivement). Conclusion. – Dans la présente étude, aucune différence significative de niveaux d’apéline et de lipocaline-2 sériques n’a été observée entre les patients atteints d’hypothyroïdie infraclinique et les sujets témoins en bonne santé. Des corrélations positives ont été trouvées, cependant, entre le niveau d’apéline sérique et l’IMC ainsi qu’entre les niveaux de lipocalin-2 sérique, l’IMC et le taux de créatinine. © 2014 Publié par Elsevier Masson SAS.

1. Introduction Thyroid hormones play a key role in the regulation of total body metabolism and adipocyte metabolism. Subclinical hypothyroidism (SCH), as an entity is defined as serum thyroxin and triiodothyronine levels ranging within normal values in the presence of elevated serum TSH levels [1]. SCH is the initial condition predisposing to thyroid disorder which causes overt hypothyroidism. Annually, about 3–18% of SCH cases progress to overt hypothyroidism [2,3]. As the precursor to hypothyroidism, SCH has been implicated also in development of metabolic syndrome. Besides metabolic syndrome, hypothyroidism is also associated with hypertension, dyslipidemia, and increased cardiovascular risk [4]. Adipocytes synthesize a large number of molecules called adipocytokines, which play a role in the pathogenesis of metabolic syndrome [5]. Apelin and lipocalin-2 are members of the adipocytokine family. Apelin was first isolated from bovine stomach extracts. This peptide is a new constituent of adipose tissue and produced by endothelial cells in many parts of the body [6]. In a study on human and murine adipocytes, apelin was reported to be released from fat cells and up-regulated by insulin [7]. Following acute intravenous injection of apelin in mice, increased glucose utilization in the skeletal muscle was observed in conjunction with a marked decrease in blood glucose. As such, apelin is highly promising for management of insulin resistance [8]. Recently, lipocalin-2 was reported to be associated with insulin resistance and obesity among humans and mice [9]. Additionally, a relationship exists between lipocalin-2 levels and metabolic syndrome, impaired lipid profile, hyperinsulinemia, hyperglycemia, and insulin resistance. In addition, elevated lipocalin-2 levels were found in patients with coronary heart disease [10]. To the best of our knowledge, there is no study on the assessment of apelin and lipocalin-2, although there are studies investigating certain members of adipokine family in patients with hypothyroidism and SCH. In addition to be an adipocytokine, Apelin is a neuropeptide and a cardiovascular peptide, that is closely associated with metabolic syndrome. Thus, the present study aimed to investigate apelin as it may be associated with SCH. In the same way, Lipocalin-2 that is closely associated with metabolic syndrome, is also associated to immune response, tumorigenesis and coronary artery disease. Thus, the present study aimed to investigate lipocalin-2 as it may be associated with the pathologies which SCH cause in the organism. We designed a protocol to determine serum levels of apelin and lipocalin-2 during SCH and to establish the degree of

association with SCH by comparing them with levels obtained from healthy subjects. 2. Patients and methods 2.1. Study group For this study, we enrolled patients aged 18 years and older with a diagnosis of subclinical hypothyroidism (TSH levels between 5–13 uIU/mL) established during their follow-up at the Bezmialem Vakif university outpatient clinic. Healthy subjects had normal thyroid function tests. Excluded from this study were patients with diabetes mellitus, malignancies, chronic kidney disease, chronic hepatic disease, any psychiatric disorders, uncontrolled hypertension, history of coronary or cerebrovascular disorder, and pregnant women. Clinical examination was carried out for all participants, and their height, body weight, and body mass index (BMI) were recorded. Body weight and height were measured to the nearest kilogram and centimeter, respectively, and BMI was calculated by the formula BMI = weight / height2. Fasting blood glucose, urea, creatinine, triglycerides, total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), serum fasting insulin level, glycosylated hemoglobin (HbA1c), whole blood count, free T3, free T4, and TSH were obtained for all subjects. Patients sera were separated from the plasma and stored at minus 80 ◦ C for later measurement of apelin and lipocalin-2 levels. The study was initiated after obtaining approval from the Ethics Committee of Bezmialem Vakif University and written consent from all participants. 2.2. Blood analysis After about 12 hours of overnight fasting, venous blood samples were drawn from the patient and control groups into gel blood collection tubes from 8:00 to 9:00 in the morning. Sera samples were separated by centrifugation for 10 minutes at a speed of 3600 rpm/minute. The homeostasis model assessment (HOMA) of insulin resistance index, a measure of insulin sensitivity, was calculated by multiplying fasting insulin concentration (␮U/mL) by fasting glucose concentration (mmol/L) / 22.5 [11]. For measurement of apelin and lipocalin-2, sera from all participants were transferred into Eppendorf tubes and stored at minus 80 ◦ C until the study was initiated. On the first study day, samples were allowed to reach room temperature and then apelin

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Table 1 Comparison of anthropometric and biochemical characteristics between the two groups groups (Mean ± SD). Group B (n = 41) Control euthyroid group

Group A (n = 39) Subclinical hypothyroid group Age (years) BMI (kg/m2 ) Apelin (ng/mL) Lipocalin-2 (ng/mL) Insulin (mU/L) HOMA-IR HbA1c (%) Fasting blood glucose (mg/dL) Total cholesterol (mg/dL) Triglycerides (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL) fT3 (pmol/L) fT4 (pmol/L) TSH (uIU/mL)

42.10 28.99 0.678 61.99 13.42 2.99 5.45 93.82 208.18 135.16 142.86 49.76 4.53 12.65 8.05

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

13.52 5.62 0.663 40.48 2.26 2.70 0.43 13.03 60.65 78.64 58.83 11.33 0.41 1.31 2.73

42.59 27.48 0.653 65.33 13.00 2.61 5.40 94.34 196.54 112.44 119.9 57.68 4.61 13.83 2.45

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

11.45 4.87 0.795 28.09 1.38 1.29 0.43 7.17 46.56 78.60 36.1 15.61 0.63 1.83 1.28

P 0.863 0.269 0.878 0.677 0.308 0.423 0.644 0.824 0.337 0.203 0.049 0.012 0.520 0.002 0.001

Mean ± SD: mean ± standard deviation; BMI: body mass index; HOMA-IR: homeostasis model assessment of insulin resistance; HbA1c: glycosylated hemoglobin; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; fT3: free triiodothyronine; fT4: free thyroxin; TSH: thyroid-stimulating hormone.

(Phoenix, Burlingame, USA) and lipocalin-2 were measured by an analyzer (Thermo ScientificTM Multiskan FC, USA) using a commercial enzyme immunoassay kit (Biovendor, Modrice, Czech Republic) according to the manufacturer’s instructions. Samples were measured in duplicate, and the average was used in the data analysis. 2.3. Statistical analysis The SPSS (Statistical Package for Social Sciences) for Windows 20.0 software package was used for statistical analyses of the study data. Student’s t test was used for comparing normally distributed parameters and Mann-Whitney U test for comparing non-normally distributed parameters, together with the use of descriptive statistical methods (mean, median, and standard deviation). Chi2 test was used for comparing data expressed in ratios. Pearson’s correlation was used to investigate correlations between variables. Statistical significance was defined at the level of 0.05 (P < 0.05).

apelin and lipocalin-2 levels (P = 0.918, P = 0.952, respectively). While HDL-C levels were significantly lower among patients compared to the control group, LDL-C levels were significantly higher (P = 0.012, and P = 0.049, respectively). No significant difference was found between groups with respect to fasting blood glucose, HOMA index, HbA1c, triglycerides, or total cholesterol (Table 1). The correlation between serum apelin and lipocalin-2 levels with hematologic, biochemical, and anthropometric parameters were investigated among patients with subclinical hypothyroidism. Significant, positive correlations were found between apelin levels and BMI and lipocalin-2 levels. Serum lipocalin-2 levels showed a positive correlation with BMI and creatinine levels (Tables 2 and 3).

Table 2 Correlations between apelin and anthropometric and biochemical characteristics of patients with subclinical hypothyroidism.

3. Results The study was conducted with a total of 80 participants divided into two groups: Group A (n = 39) comprised patients with subclinical hypothyroidism (females, n = 34), and Group B (n = 41 controls,) was made up of healthy volunteers (females, n = 38). The two groups were comparable with respect to gender, age, and body mass index (BMI), with no statistically significant inter-group difference (P = 0.412, P = 0.863, and P = 0.269, respectively) (Table 1). The groups had no significant differences in terms of apelin and lipocalin-2 levels (P = 0.87 and P = 0.67, respectively) (Table 1). A vast majority of subjects in both groups were females; however, results remained the same when males were excluded. There was also no significant difference between female groups (females with SCH and controls) in terms of both

Age BMI HOMA-IR Lipocalin-2 Total cholesterol Triglycerides LDL-C HDL-C Creatinine fT3 fT4 TSH

r

P value

0.039 0.345 0.008 0.351 –0.142 –0.010 –0.076 –0.097 0.010 –0.247 0.76 –0.100

0.816 0.034 0.960 0.030 0.397 0.955 0.660 0.567 0.953 0.136 0.656 0.550

BMI: body mass index; HOMA-IR: homeostasis model assessment of insulin resistance; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; fT3: free triiodothyronine; fT4: free thyroxin; TSH: thyroid-stimulating hormone.

M. Zorlu et al. / Annales d’Endocrinologie 75 (2014) 10–14 Table 3 Correlations between lipocalin-2 and anthropometric and biochemical characteristics of patients with subclinical hypothyroidism.

Age BMI HOMA-IR Apelin Total cholesterol Triglycerides LDL-C HDL-C Creatinine fT3 fT4 TSH

r

P value

0.147 0.493 0.063 0.351 –0.093 0.063 –0.074 –0.042 0.363 –0.101 –0.51 –0.093

0.377 0.002 0.754 0.030 0.579 0.710 0.670 0.804 0.025 0.547 0.765 0.579

BMI: body mass index; HOMA-IR: homeostasis model assessment of insulin resistance; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; fT3: free triiodothyronine; fT4: free thyroxin; TSH: thyroid-stimulating hormone.

4. Discussion We obtained serum levels of apelin and lipocalin-2 among patients with subclinical hypothyroidism and in order to determine associations between these adipokines and anthropometric and metabolic characteristics. For this purpose, the study protocol targeted enrollment of patients who had matching demographics, anthropometric characteristics, and comorbidities in order to reduce confounding external factors as much as possible. The patients and the controls exhibited no significant difference in age, BMI, HOMA-IR, or HbA1c. Our literature search did not identify any study which investigated apelin and lipocalin-2 levels among patients with subclinical hypothyroidism; however, a limited number of studies exist which examined certain adipokines in patients with subclinical hypothyroidism and hypothyroidism. In our study, no difference was observed between patients with subclinical hypothyroidism and the control group in terms of serum apelin levels. Aksoy et al. examined the levels of resistin (a member of adipokine family) among 36 female patients with subclinical hypothyroidism and 27 healthy female controls before and after 6 months of treatment with L-thyroxin. They found no difference before or after treatment [12]. C ¸ ınar et al. compared vaspin levels among 27 patients with overt hypothyroidism (33 patients with subclinical hypothyroidism and 41 healthy controls) and found no difference between them [13]. These results are consistent with ours. The aforementioned results might also suggest that there is no relationship between some of the members of adipokine family with the metabolic syndrome and/or with subclinical hypothyroidism. On the other hand, some study results contradict ours. Jing et al., comparing levels of visfatin (an adipokine) among hypothyroid and hyperthyroid patients and euthyroid subjects, found significantly higher plasma visfatin levels in their hypothyroidic and hyperthyroidic patients [14]. They repeated the same study using an animal model of Wistar rats and showed that serum visfatin levels were significantly higher in

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hypothyroid animals compared to euthyroidic animals [14]. The significant difference might have resulted from the absence of subjects with subclinical hypothyroidism and inclusion of patients with overt hypothyroidism. Conversely, a similar relationship with subclinical hypothyroidism may be present due to the fact that subclinical hypothyroidism is a predisposing condition and a milder form of hypothyroidism. Apelin and lipocalin-2 levels were also shown to be higher in obese individuals and in persons with a higher degree of insulin resistance [9,15]. Our study showed no difference between groups with respect to BMI or HOMA index, suggesting that these results are more specific for subclinical hypothyroidism. When compared with hematological, biochemical, and anthropometric parameters among patients with subclinical hypothyroidism, apelin levels showed significant positive correlations with BMI and lipocalin-2 levels (P = 0.034 and P = 0.030, respectively), and serum lipocalin-2 levels showed significant positive correlations with BMI and creatinine levels (P = 0.002 and P = 0.025, respectively). Our literature search yielded one study showing a possible association of lipocalin-2 level with urinary output and renal failure in a setting of acute renal failure and among transplant patients [16]. Further larger scale studies on lipocalin-2 might provide a clearer picture of its association with renal failure. Different results obtained in previous studies might have resulted from their limited number of cases and insufficiently matched comparison groups. The positive correlation between BMI and serum apelin and lipocalin-2 levels could indicate failure to match both groups for BMI, wrongly leading to a false overestimation of the association with subclinical hypothyroidism. The present study included only patients with idiopathic hypothyroidism, most probably associated to Hashimoto’s thyroiditis. Moreover, in our study, the number of overweight subclinical hypothyroidism patients was well-matched with similarly heavy controls in order to provide a better equalization of the two groups and to minimize the possibility of a false result. In conclusion, the patients with subclinical hypothyroidism and healthy controls did not differ significantly with respect to serum apelin or lipocalin-2 levels. Positive association of both serum apelin and lipocalin-2 with BMI could be interpreted as an important indicator of weight gain. In addition, the positive correlation of lipocalin-2 with creatinine suggest that lipocalin-2 could be a marker for progression of renal insufficiency. Further larger studies are needed to clarify these issues. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements The authors appreciate the contributions and editorial assistance of S. Delacroix.

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