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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-0562
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 10 5765-5768
Copyright © 2005 by The Endocrine Society

BRIEF REPORT

Changes in Plasma Concentrations of Osteoprotegerin before and after Levothyroxine Replacement Therapy in Hypothyroid Patients

Xiang Guang-da, Sun Hui-ling, Cheng Zhi-song and Zhao Lin-shuang

Department of Endocrinology, Wuhan General Hospital of Guangzhou Command, Wuhan 430070, Hubei Province, People’s Republic of China

Address all correspondence and requests for reprints to: Xiang Guang-da, M.D., Ph.D., Department of Endocrinology, Wuhan General Hospital of Guangzhou Command, Wuluo Road 627, Wuhan 430070, Hubei Province, People’s Republic of China. E-mail: Guangda64{at}hotmail.com.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Recent study has shown that overt hypothyroidism (oHT) is associated with increased plasma osteoprotegerin (OPG) levels.

Objective: Our objective was to examine the plasma OPG level alteration before and after levothyroxine (L-T4) treatment in oHT and subclinical hypothyroidism (sHT).

Patients: The study subjects included oHT and sHT patients and healthy individuals (20 subjects in each group).

Methods: All patients were given L-T4 therapy to maintain a euthyroid state. Plasma OPG concentration was measured in duplicate by a sandwich ELISA.

Results: Plasma OPG levels in oHT and sHT before treatment were significantly higher than levels in controls (P < 0.01). After normalization of thyroid function, OPG levels in both groups decreased markedly (P < 0.01). The absolute changes in OPG showed a significant positive correlation with the changes in TSH (P < 0.05) and negative correlation with the changes in endothelium-dependent arterial dilation (P < 0.01) in hypothyroid patients during the course of treatment.

Conclusion: OPG may be involved in the development of vascular dysfunction in hypothyroid patients.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
OSTEOPROTEGERIN (OPG) HAS been identified as an inhibitor of bone resorption. This inhibition is mediated through OPG’s binding and neutralization of the receptor activator of nuclear factor-{kappa}B ligand. Recently, some studies have suggested that OPG also acts as an important regulatory molecule in the vasculature (1, 2, 3, 4). Overt hypothyroidism (oHT) and subclinical hypothyroidism (sHT) are associated with increased risk for cardiovascular disease (5, 6, 7). More recently, one study showed that plasma OPG increases significantly and decreases markedly with levothyroxine (L-T4) therapy in patients with oHT (8). To date, no data are available on the relationship between OPG levels and sHT. The purpose of this study was to investigate the alteration of plasma OPG concentrations before and after L-T4 therapy in oHT and sHT.


   Subjects and Methods
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 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

From January 2002 to January 2003, 20 women with oHT, 20 women with sHT, and 20 healthy women with euthyroid state were studied. All patients were newly diagnosed with Hashimoto’s thyroiditis and were positive for both antithyroid peroxidase antibodies (TPO-Ab) and antithyroglobulin antibodies (Tg-Ab). The diagnosis of oHT was established on the basis of suppression of serum free T4 (FT4) and free T3 (FT3) below the normal lower limit and elevation of serum TSH above the normal upper limit. sHT was defined as elevated TSH levels and normal FT3 and FT4 values. All patients were premenopausal, with regular menses. Obese subjects [body mass index (BMI) > 30 kg/m2], smokers, and those with hypertension, clinical detectable coronary artery disease, and other diseases were excluded from the study. Also, no patient was taking any drugs, such as oral contraceptives. All subjects gave informed consent. The study protocol was in agreement with the guidelines of the ethics committee at our hospital.

Patients were then given L-T4 therapy individually to maintain all of thyroid function near or within the respective normal ranges. All patients were measured for endothelium-dependent arterial dilation (EDD) as well as biochemical markers before and after at least 0.5 yr (6–8 months) of normalization of thyroid function (mean therapy period, 113 months for oHT and 102 months for sHT).

Methods

Venous blood samples were drawn after a 12-h overnight fast. The plasma concentrations of OPG were measured in EDTA-plasma samples by a commercially available kit (R&D Systems, Minneapolis, MN). This assay is a sandwich ELISA, using a mouse antihuman OPG as capture antibody and a biotinylated goat antihuman OPG for detection. Recombinant human OPG was used for calibration, and the range of the assay was 62.5–4000 pg/ml. Plasma samples were diluted 1/3 and measured in duplicate, and the results were averaged. The intraassay coefficient of variation was 3–6.5%.

Measurement of serum lipids, lipoproteins, and other parameters, serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL), triglyceride, and high-density lipoprotein cholesterol (HDL), were measured enzymatically. Apolipoprotein A1 (ApoA1) and ApoB were measured by immunoturbidimetry. Serum lipoprotein (a) [Lp(a)] was measured by an ELISA. Fasting serum glucose (FBG) was measured by a glucose oxidase procedure. Creatinine kinase (CK), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatinine were measured enzymatically. The concentrations of FT3 and FT4 were measured by RIA, and TSH was determined with an ultrasensitive immunoradiometric assay. Tg-Ab was measured by specific immunoradiometric assay, and TPO-Ab was measured by specific RIA. The intraassay coefficients of variation for these assays were 1–2% (TC, HDL, FBG, CK, AST, LDH, FT3, FT4, TSH, and creatinine), 2–3% (LDL), 2–4% (ApoA1 and ApoB), and 4–7% [Lp(a), Tg-Ab, and TPO-Ab].

The vascular studies of the brachial artery, including baseline vessel size, baseline flow, EDD, and glyceryltrinitrate-mediated endothelium-independent arterial dilation (EID), were performed noninvasively, as described by us previously (9, 10).

Statistical methods

Data are reported as the mean ± SD. The difference in each parameter between before and after treatment was compared using the Student’s t test (two-tailed) for paired data, and the difference between patients and controls was compared by the Student’s unpaired t test. Correlations between the changes of OPG and the changes of clinical and biochemical characteristics and results of brachial artery studies in hypothyroidism during treatment were determined by Spearman’s analysis. Lp(a) concentrations were log-transformed before analysis.


   Results
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 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
The data confirm that these three groups had oHT and sHT before treatment and were euthyroid after treatment. In oHT, compared with control, TC, LDL, and Lp(a) levels were significantly higher (P < 0.05) and decreased significantly after treatment (P < 0.05). Also, there were similar changes of LDL and Lp(a) levels in sHT (P < 0.05) (Table 1Go).


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  		TABLE 1. Clinical and biochemical characteristics and results of brachial artery studies in hypothyroidism before and after therapy as well as control group

 
OPG levels in oHT and sHT before treatment were significantly higher than that in controls (P < 0.01). After treatment, OPG levels in both groups decreased markedly (P < 0.01), and there was no significant difference between patients at the end of therapy and controls (P > 0.05). As shown in Fig. 1Go, OPG changes of most patients showed a marked decrease, and only one patient with oHT and two with sHT showed a slight increase during the course of treatment.



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  		FIG. 1. Changes in OPG levels before and after treatment in oHT and sHT.

 
At baseline, the EDD among subjects with sHT and oHT were significantly lower than that in controls (P < 0.01), and the EDD among subjects with oHT was significantly lower than that in subjects with sHT (P < 0.01). After treatment, the EDD among subjects with sHT and oHT were significantly higher than those before treatment (P < 0.01), and there was no significant difference between patients at the end of therapy and controls (P > 0.05) (Table 1Go).

The absolute changes in OPG showed significant correlation with the changes in TSH (P < 0.05) and in EDD (P < 0.01) in hypothyroid patients during the course of treatment.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Recently, one study showed that plasma OPG increases significantly and decreases to normal range after normal thyroid function with L-T4 therapy in patients with oHT (8). In the present study, we also found similar changes in plasma OPG levels in both oHT and sHT patients during the course of L-T4 therapy. As far as we know, this is the first report on the relation between plasma OPG and sHT.

Many studies showed that oHT, even sHT, are associated with increased risk for cardiovascular disease (5, 6, 7). Recently, some studies suggested that oHT (11) and sHT (12) are associated with impaired EDD, and L-T4 therapy improves EDD. Here, we found similar results as well. In a prospective study of almost 500 women, high OPG values were associated with an increased cardiovascular mortality (13). In another investigation, the authors found an association between OPG levels and the presence and severity of coronary artery disease in subjects undergoing coronary arteriography (14). More recently, Nagasaki et al. (8) found the change in plasma OPG levels during L-T4 therapy was associated in a negative fashion with baseline plasma von Willebrand factor in oHT. In the present study, during the treatment course, the absolute changes in OPG showed significant correlation with the changes in EDD. Therefore, we speculate that the elevated OPG levels may be associated with the increased risk of vascular diseases in hypothyroid patients.

Several studies support a role of OPG in vascular homeostasis. Thus, OPG is abundantly expressed in the media of large arteries and in atherosclerotic plaques (1) as well as in vascular muscle cells (3). Furthermore, OPG has been demonstrated to act as a survival factor for endothelial cells (4), and OPG knockout mice have calcifications and aneurysms in the large arteries (2). The development of arterial calcification was completely prevented by crossbreeding OPG-deficient mice with OPG transgenic mice in which the transgene had been delivered prenatally. Thus, increased OPG levels may represent an (incomplete) defense mechanism against other factors that promote arterial calcification, atherosclerosis, and other forms of vascular damage.

Various mechanisms not related to the vascular damage should be considered. In the human thyroid follicular cell line XTC and in primary human thyroid follicular cells, OPG mRNA levels and protein secretion were up-regulated by TSH (15). In the present study, the changes in OPG showed significant correlation with the changes in TSH during L-T4 therapy. These data can partially explain the elevated plasma OPG levels in hypothyroid patients. However, one study showed that hyperthyroid patients have serum OPG concentrations significantly higher in comparison with euthyroid subjects, in relation to thyroid hormone excess and high bone turnover. Medical treatment of hyperthyroidism normalizes serum OPG levels in temporal relationship with the normalization of bone metabolism markers (16). Another study revealed that OPG mRNA expression levels were stimulated by T3 in mature MC3T3-E1 cells but not in preosteoblastic MC3T3-E1 cells or in ST2 cells (17). These results suggest the possibility that altered thyroid states have different effects on OPG expression, with increased plasma OPG mainly produced by high bone turnover in hyperthyroidism. Also, there is evidence to suggest that thyroid autoimmunity may modulate the local and systemic OPG production (15, 16). OPG mRNA levels were three times more abundant in surgical thyroid specimens of Graves’ disease compared with other thyroid diseases (15). In the present study, our patients suffered from Hashimoto’s thyroiditis. Thyroid autoimmunity may contribute to the elevated plasma OPG values in the present study.

In addition, in accordance with a recent study (8), serum creatinine did not change significantly before and after L-T4 treatment. These data may negate the possibility, although not completely, that decreased clearance of OPG from serum in the hypothyroid state might increase serum OPG levels. In the present study, there were not any correlations between the absolute changes of OPG levels and the changes of blood pressure, BMI, AST, CK, LDH, and blood lipids before and after treatment in hypothyroid patients, which are in good agreement with the previous studies (13, 18). We also found that the lipid changes including Lp(a), TC, and LDL, were consistent with previously published data (19).

In conclusion, we found increased levels of OPG from oHT and sHT patients, which were close to those of controls after normalization of thyroid function. This finding supports the growing concept that OPG may be involved in the development of vascular dysfunction in hypothyroid patients.


   Footnotes
 
First Published Online July 26, 2005

Abbreviations: ApoA1, Apolipoprotein A1; AST, aspartate aminotransferase; BMI, body mass index; CK, creatinine kinase; EDD, endothelium-dependent arterial dilation; EID, endothelium-independent arterial dilation; FBG, fasting blood glucose; FT4, free T4; HDL, high-density lipoprotein cholesterol; LDH, lactate dehydrogenase; LDL, low-density lipoprotein cholesterol; Lp(a), lipoprotein (a); L-T4, levothyroxine; oHT, overt hypothyroidism; OPG, osteoprotegerin; sHT, subclinical hypothyroidism; TC, total cholesterol; Tg-Ab, antithyroglobulin antibodies; TPO, antithyroid peroxidase antibodies.

Received March 14, 2005.

Accepted July 18, 2005.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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