Can Serum Osteocalcin Level be Used as a Marker to Assess Bone Remodeling Status in Hyperthyroidism?

Aims: The aim of this study was to estimate the serum osteocalcin levels before and during treatment in hyperthyroidism and to compare with healthy controls. Study Case control study. Methodology: Twenty five newly diagnosed hyperthyroid patients, Twenty five patients who are euthyroid during treatment and Twenty five healthy controls in the age group of 20-50yrs were enrolled in this study. Serum T3, T4, TSH were assayed by electrochemiluminescence and serum Osteocalcin was estimated by ELISA. Kruskal wallis test was used to compare T3, T4, TSH, osteocalcin and calcium levels among three groups. Post Hoc test was used for multiple comparisons between two groups. Results: Using Kruskal wallis test, a significant difference for T3, T4, TSH and osteocalcin values was observed among the different groups (p <0.001). Post Hoc test for multiple comparisons between two groups revealed that osteocalcin levels were significantly high in newly diagnosed hyperthyroid patients compared to controls (p=0.02) indicating high bone turnover. There was also a significant difference in osteocalcin levels between newly diagnosed hyperthyroid patients and patients who are euthyroid on treatment (p= 0.04) indicating values decreased on treatment. ROC analysis showed serum osteocalcin is a good marker for assessing bone remodeling status in hyperthyroidism. (Area under curve= 86.6 and p value = 0.001) Conclusion: Serum osteocalcin can be used to assess bone remodeling status in newly diagnosed hyperthyroidism and to identify residual bone resorption in the euthyroid state during treatment. Area under the curve was similar for both osteocalcin and calcium. Osteocalcin levels were tending towards normal although not completely falling within the range established for the control group where the median value in euthyroid state, for osteocalcin was higher 0.1 ng/ml with an interquartile range of ( 0.001-2.20) as compared to 0.001 ng/ml with an interquartile range of (0.001-0.15) in controls. Considering the range established in both these groups, this study throws light on the fact that values of osteocalcin >0.15 ng/ml and < 2.20 ng/ml probably still indicate a certain degree of bone resorption although not statistically insignificant.


INTRODUCTION
Hyperthyroidism is an endocrinological disorder that exhibits low serum TSH levels and elevated levels of the thyroid hormones. Hyperthyroidism includes diseases that are a subset of thyrotoxicosis, caused by excess synthesis and secretion of thyroid hormone. Graves disease is an autoimmune thyroid disease in which anti TSH receptor autoantibodies cause hyperthyroidism. Hyperthyroidism is sometimes caused by paraneoplastic disease. Because of this, in these cases normalizing the thyroid function with antithyroid drugs can not improve prognosis [1].
Hyperthyroidism is accompanied by osteoporosis with increased rate of bone formation and bone resorption caused from direct stimulation of bone cells by increased level of thyroid hormones. The severity of hyperthyroidism is directly related to the alteration in the levels of biochemical markers of bone turnover and bone loss [2]. Studies have shown that euthyroid state will only partially reduce hyperthyroid osteopenia with a bone mass recovery during 6-9 months of effective treatment [3].
Osteocalcin is a non collagenous protein found in bone and dentin. It is secreted solely by osteoblasts and is pro-osteoblastic or bone building by nature. It is implicated in bone mineralization and calcium ion homeostasis [4].
Calcium is one of the abundant extracellular cations in the body and it plays an important role in bone formation. Thyroid hormones stimulate bone resorption directly there by increasing the serum calcium levels and suppressing parathyroid hormone [5].
Osteocalcin being a bone formation marker is supposed to increase in hyperthyroidism [2]. Therefore, this study was done to show the correlation between serum osteocalcin levels and serum calcium in hyperthyroidism which may in turn suggest the need for calcium supplementation to prevent osteoporosis in these patients. All subjects were in the age group of 20-50 years. Written informed consent was taken from both cases and controls. Study protocol was approved by Ethical committee of the Institute. Patients of age group >50 yrs, postmenopausal women, history of osteoporosis, fractures, hepatic dysfunction, parathyroid dysfunction, renal disease, malignancies and patients who were on calcium supplements, vitamin D supplements, steroids and thiazides were excluded from the study. About 2 ml of venous blood was collected. Serum was separated by centrifugation and stored at -20°C until the estimation of biochemical parameters.

RESULTS
The results of all variables were expressed as median values as our data did not follow normal Gaussian distribution. Kruskal wallis test was used to compare T3, T4, TSH, osteocalcin and calcium levels among three groups. Post Hoc test was used for multiple comparisons between two groups. Results were computed using Median values for T3 and T4 in newly diagnosed hyperthyroid patients were found to be increased when compared to controls and patients on treatment in the euthyroid state. Patients on treatment achieved T3 and T4 values which is almost normal.
Median values for TSH in newly diagnosed hyperthyroid patients were low compared to controls and patients on treatment with a statistically significant difference of p=0.001.
Osteocalcin levels were elevated in hyperthyroid state compared to controls. In those patients who are euthyroid during treatment, serum osteocalcin levels decreased considerably but were found to be marginally high as compared to the control group.
Median values obtained for calcium in group 1 was 11.33(10.6-12.21), group 3 was 11.07 (10-11.96) and in controls 10.3 (9.2-11.19). This indicates that compared to the controls the calcium concentration was highest in the serum in newly diagnosed hyperthyroid patients and tending towards normal in patients who are euthyroid during treatment but higher than that of controls.

TSH-
There was a significant difference in TSH values between newly diagnosed hyperthyroid patients and controls as also between newly diagnosed hyperthyroid patients and patients who are euthyroid during treatment.
There was a moderate significant difference between controls and patients who are euthyroid during treatment which indicates TSH values are increasingly tending towards normal.
There was a significant difference in osteocalcin levels between newly diagnosed hyperthyroid patients and controls indicating hyperthyroid patients have a high bone turnover.
There was a significant difference in osteocalcin levels between newly diagnosed hyperthyroid patients and patients who are euthyroid on treatment indicating the values decreased on treatment. There was no significant difference between patients who are euthyroid on treatment and controls.
There was a significant difference in calcium levels between newly diagnosed hyperthyroid patients and controls. There was no significant difference in calcium levels between newly diagnosed hyperthyroid patients and patients who are euthyroid on treatment and also between patients who are euthyroid on treatment and controls.
There was a positive correlation between T3 and osteocalcin in newly diagnosed hyperthyroid patients with a correlation coefficient of 0.537.

DISCUSSION
Hyperthyroidism is characterized by accelerated bone turnover, caused from direct stimulation of bone cells by increased thyroid hormones. The cycle of bone remodeling is shortened to almost 50% (from 200 to 113 days), and the proportions between bone formation and bone resorption are disturbed. Those with a history of untreated thyrotoxicosis extending for more than a year, may have severe and premature osteoporosis [11].
Triiodothyronine (T3), is responsible for major actions of thyroid hormones. T3 binds to nuclear receptors that regulate gene transcription via interaction with thyroid hormone response elements of specific genes [12].
T3 regulates the chondrogenesis bone mineralization, angiogenesis and bone matrix formation. T3 stimulates the IL-6 and IL-8, intensifies the effects of IL-1 and IL-6, augments the synthesis of osteocalcin, collagen type 1, increases proliferation, differentiation and apoptosis of osteoblasts [13,14]. Also, TSH deficiency could be partly responsible for the skeletal loss seen in thyrotoxicosis, because it has been proposed that TSH may be a direct negative regulator of bone turnover acting via the TSH receptor on both osteoblasts and osteoclasts, as TSH inhibits osteoclast formation and survival. It also inhibits differentiation of osteoblasts [15].
A similar study conducted by Barsal et al. [16] showed that there is increase in bone formation marker Osteocalcin and ALP as well as bone resorption marker, urinary calcium/ creatinine levels in Hyperthyroidism. These markers failed to normalize even after attaining euthyroidism. Hence he concluded that hyperthyroid patients have a high bone turnover of formation and resorption even after attainment of euthyroidism and osteocalcin is a sensitive marker in documenting bone remodeling during treatment of Hyperthyroidism [17].
Another study conducted by Siddiqi et al observed a fall of the bone resorption markers urinary collagen pyridinoline and deoxypyridinoline and bone formation markers osteocalcin and bone ALP after 4-8 weeks of antithyroid treatment [18,19].
The accelerated bone remodeling cycle in hyperthyroid state results in increased bone resorption and a subsequent increased release of calcium into systemic circulation [20].
High levels of serum calcium inhibit parathyroid hormone (PTH) secretion and create a negative calcium balance through prolonged urinary and fecal losses. Decreased PTH secretion causes hypercalciuria as a protective mechanism against hypercalcemia [20].
With low PTH, Vitamin D is not converted into its active form, leading to low gastrointestinal calcium and phosphorous absorption and resultant fecal calcium losses [20].
Calcium balance which was negative during the hyperthyroid status was converted to positive soon after attainment of euthyroidism which is due to healing of the metabolic bone disease and increased calcium deposition in to the bone [21,22].
Calcium levels in newly diagnosed hyperthyroid patients was in upper normal range when compared to lower normal range of controls. There was no significant difference in calcium levels in newly diagnosed hyperthyroid patients and those who are euthyroid during treatment as also between patients who are euthyroid during treatment and controls.
Osteocalcin levels in newly diagnosed hyperthyroid patients is significantly high compared to patients who are euthyroid during treatment and controls. During treatment, osteocalcin levels showed a significant fall but failed to achieve the range established for controls indicating a probable phase of residual bone resorption even though the patients were euthyroid. Therefore, estimating both Serum Osteocalcin and serum Calcium together in hyperthyroidism will reflect the bone remodeling status better.

CONCLUSION
Antithyroid drugs can help the patient to achieve an euthyroid state but the residual bone loss can be identified and treated only by monitoring both serum calcium levels and the levels of bone formation markers like serum osteocalcin. Such patients can be supplemented with calcium, vitamin D and bisphosphonates to improve bone mineralization and prevent osteoporosis.