Study of copper and ceruloplasmin in hypothyroidism and Grave's hyperthyroidism as compared to euthyroidism: Hospital-based case–control study Bhat VG, Patra R, K. Raju D S, Mohandas G V, Rao N L,

Users Online: 1123

ORIGINAL ARTICLE

Ahead of print publication

Study of copper and ceruloplasmin in hypothyroidism and Grave's hyperthyroidism as compared to euthyroidism: Hospital-based case–control study

Vinayak Ganesh Bhat¹, Rajkumar Patra², D S. S K. Raju¹, GV Mohandas³, N Lakshmana Rao⁴
¹ Department of Biochemistry, Maharajah's Institute of Medical Sciences, Vizianagaram, Andhra Pradesh, India
² Department of Ophthalmology, Maharajah's Institute of Medical Sciences, Vizianagaram, Andhra Pradesh, India
³ Department of Anatomy, Maharajah's Institute of Medical Sciences, Vizianagaram, Andhra Pradesh, India
⁴ Department of Community Medicine, Maharajah's Institute of Medical Sciences, Vizianagaram, Andhra Pradesh, India

Click here for correspondence address and email

Date of Submission	24-Nov-2022
Date of Decision	30-Nov-2022
Date of Acceptance	26-Jan-2023
Date of Web Publication	10-May-2023

Abstract

Background: Hypothyroidism was highly prevalent in India, with 10.95% affected patients as compared with only 2% in the United Kingdom and 4·6% in the USA. The prevalence of Hyperthyroidism was found to be 1.6%. Copper has an essential role in thyroid metabolism, thyroid hormone production, and absorption. Copper is an essential part of thyroxine (T4) hormone and thyroid-stimulating hormone (TSH) synthesis.
Objectives: This study evaluated the status of serum copper and ceruloplasmin levels in hypothyroidism and Grave's hyperthyroidism in comparison with euthyroid participants.
Materials and Methods: This was a hospital-based case–control study that involved 100 cases of hypothyroidism and 60 cases of Grave's hyperthyroidism compared with 100 euthyroid participants aged 15–60 years. After detailed case history and examination, the blood sample was collected and analyzed the same day for FreeT3, FreeT4, TSH, serum copper, and ceruloplasmin levels.
Results: Compared to euthyroid participants, patients with clinical hypothyroidism had statistically significant (P < 0.05) lower levels of serum copper and ceruloplasmin. The scenario was the opposite in the case of Grave's hyperthyroidism patients. Our study found statistically significant (P < 0.05) higher serum copper and ceruloplasmin levels in Grave's hyperthyroidism patients.
Conclusion: Our study found serum copper levels directly affected the thyroid profile and vice versa. The deficiency of one parameter affected the other. Patients with hypothyroidism and hyperthyroidism should be routinely screened for serum copper and ceruloplasmin levels for more effective thyroid treatment.

Keywords: Ceruloplasmin, copper, cross-sectional study, Grave's hyperthyroidism, hypothyroidism, thyroid profile

How to cite this URL:
Bhat VG, Patra R, K. Raju D S, Mohandas G V, Rao N L. Study of copper and ceruloplasmin in hypothyroidism and Grave's hyperthyroidism as compared to euthyroidism: Hospital-based case–control study. MRIMS J Health Sci [Epub ahead of print] [cited 2023 May 29]. Available from: http://www.mrimsjournal.com/preprintarticle.asp?id=376664

Introduction

Thyroid disease is widespread in the world. India being no exception, have a significant burden of thyroid diseases, among which hypothyroidism is prevalent.^[1] Hypothyroidism is a clinical condition that occurs due to the low level of circulating thyroid hormones. It can easily be diagnosed and treatable. It often presents to the hospital with nonspecific symptoms and is hence diagnosed accurately by measuring the thyroid hormone levels in the blood. Thyroid hormones are vital for the regulation of the human body's metabolism. Hypothyroidism, when left untreated, can complicate health by myxedema, cardiac failure, coma, and death.

Hypothyroidism is highly prevalent in the Indian population. 1 in 10 people in the country was diagnosed with hypothyroidism. As per a study, more than 200 million people have thyroid disease worldwide. The prevalence of hypothyroidism in India is 10.95%^[1] compared with only 2% in the United Kingdom and 4·6% in the USA. The prevalence of hyperthyroidism was found to be 1.6%.^[2] The prevalence of thyroid disease increases with age and is more common in women. The prevalence of subclinical hypothyroidism is higher than clinical hypothyroidism, which goes undiagnosed most of the time.

Copper is the third-most abundant mineral seen in humans. Copper plays an essential role in thyroid metabolism, thyroid hormone production, and absorption. Copper is an essential part of thyroxine (T4) hormone and thyroid-stimulating hormone (TSH) synthesis.^[3],[4] On the other hand, the thyroid hormone regulates serum copper levels, through the regulation of ceruloplasmin synthesis.^[5],[6],[7] Hence, an intricate relationship exists between copper and thyroid hormone.

The objective of this case–control study was to evaluate the status of serum copper and ceruloplasmin levels in hypothyroidism and Grave's hyperthyroidism participants keeping euthyroid patients in control. There was no study done in India till today to understand this intricate association. This was the purpose of the current study.

Materials and Methods

This was a hospital-based case–control study. All the patients with TSH hormone levels >4.2 mIU/dl with free T4 levels 0.8 ng/dl or free T3 levels <210 pg/dl or both, with or without symptoms and signs, were taken as cases of hypothyroidism. All the patients with TSH hormone levels <0.4 mIU/dl with Free T4 levels >2.7 ng/dl or Free T3 levels >440 pg/dl or both, with signs of Grave's ophthalmopathy, were taken as cases of Grave's hyperthyroidism. Age- and gender-matched participants from the same rural villages and urban areas of the district were taken as control. TSH, Free T3, and Free T4 levels were checked for confirmation of euthyroidism. Detailed dietary histories were taken in both cases and controls and matched. All the patients were newly diagnosed with hypothyroidism and Grave's hyperthyroidism and were diagnosed first time in our hospital. All the patients were untreated cases. Sources of cases and controls were the participants who attended OPD of the Institution during the study period. Convenience sampling was done over a period of 1.5 years. All the patients who visited the institution during the period with our criteria were enrolled in the study. The study included 100 newly diagnosed cases of hypothyroidism and 60 newly diagnosed cases of Grave's hyperthyroidism in comparison with 100 euthyroid participants aged 15–60 years who attended the OPD of the institution for 1.5 years from January 15, 2021, to July 15, 2022. The study protocol was presented to the ethical committee of the institution. It was approved by the institutional ethical committee on January 7, 2021, by letter number IEC/105/21. Written informed consent was taken from all the study participants.

Exclusion criteria included participants on copper supplements and participants on any medication that can alter thyroid status or copper status in blood. Exclusion Criteria also included Pregnant, Menopause or Lactating women, Subjects with Acute or Chronic illness, Liver or Renal disorders, Thyroid malignancies, Subjects with intake of Oral contraceptive pills.

Potential bias was limited in the study. Patients were picked up based on the disease criteria. The primary researchers were a trained ophthalmologist and a trained biochemist. No other ophthalmologist or biochemist was involved in patient data collection. Diagnostic bias was limited as the same set of instruments, and testing kits were used for all patients. Testings were done by the primary Researcher.

Detailed clinical history was taken from all the study participants who participated in the study, along with detailed dietic history. A general physical examination was done, including height, weight, blood pressure measurement, and pulse measurement, was done and recorded in a structured protocol format. Grave's hyperthyroidism participants were examined in detail for exophthalmos in the department of ophthalmology and recorded in detail. After obtaining Informed consent from the subject, 5 mL of the venous blood was taken from all participants under aseptic conditions. The serum tube was centrifuged after adequate clotting. The serum was separated in the new test tube and aliquoted. The serum was tested for thyroid profile (Free T3, Free T4, and TSH), serum levels of copper and ceruloplasmin. The aliquoted sample was stored at −20°C. The following instruments and methods were used for analyzing the parameters. Free T3, Free T4, TSH and Ceruloplasmin were analyzed by Chemiluminescence Immunoassay (CLIA) method in SNIBE MAGLUMI 800 analyzer. Serum copper was analyzed by the Spectrophotometric method using Di-Br-PAESA reagent in ERBA EM200 analyzer.

IBM SPSS Statistics Version 21 Software which was developed by IBM Software company was used to analyze the data. was used to analyze the data. Quantitative variables were expressed in mean and standard deviations (SDs). ANOVA with post hoc analysis was used for the comparisons between three groups and multiple comparisons. Karl–Pearson correlation coefficient was used to explore the association between study variables. For all statistical analyses, P < 0.05 was considered statistically significant.

Results

Age and gender in the study groups were not significantly different between different study groups, as shown in [Table 1]. Mean ± SD of FT3 and FT4 were significantly lower in hypothyroidism (P < 0.05) and significantly higher in Grave's Hyperthyroidism (P < 0.05) in comparison with euthyroid participants. [Table 2] analyzes and compares the mean ± SD of serum copper and ceruloplasmin levels in various groups. The mean ± SD of serum copper was found to be 47.38 ± 17.69 μg/dl in the hypothyroid group and 164.64 ± 14.98 μg/dl in the hyperthyroid group compared to 105.43 ± 20.17 μg/dl in controls. A strong association was established during comparison (P < 0.05). Similar results were found with serum ceruloplasmin. Mean ± SD of serum ceruloplasmin was found to be 10.44 ± 1.69 mg/dl in the hypothyroid group and 70.85 ± 3.73 mg/dl in the hyperthyroid group compared to 49.17 ± 3.06 mg/dl in controls. A strong association was established during comparison (P < 0.05). [Figure 1] and [Figure 2] show Pearson correlation within various study groups. The hypothyroidism group showed a significant association between copper and TSH (P = 0.016). The relationship between FT3 and TSH was also statistically significant (P = 0.075). Similarly, the hyperthyroid group also showed a significant association between FT3 and ceruloplasmin (P = 0.022). The relationship between copper and FT3 (P = 0.074) was also statistically significant.

Figure 1: Serum copper with TSH in the hypothyroidism group. TSH: Thyroid-stimulating hormone

Click here to view

Figure 2: Ceruloplasmin with free T3 in the hyperthyroidism group

Click here to view

Table 1: Distribution of subjects based on age

Click here to view

Table 2: Mean, standard deviation, and P value of serum copper and ceruloplasmin in groups

Click here to view

[Figure 1] and [Figure 2] show the Scatter Diagram of various Parameters in different study subjects:

Discussion

Thyroid disorders are highly prevalent in India. 1% of the general population is affected by hypothyroidism and about 5% of the population is over the age of 60 years.^[8] India has 42 million people with thyroid disorders, per the national workshop in Chennai on June 5, 2014. Hypothyroidism is the most common thyroid disorder affecting one in ten adults in India. Its prevalence in India was 11% compared with the U. K (2%) and the USA (4.6%), as per Bagcchi.^[9]

Our age and gender of participants match within the three groups of our study and also with other major studies done in India. In our study, the mean ± SD of serum copper in clinical hypothyroidism was found to be 47.38 ± 17.69 μg/dl, and Grave's hyperthyroidism was 164.64 ± 14.98 μg/dl in comparison with euthyroid participants which was found to be 105.43 ± 20.17 μg/dl. The copper deficiency in patients with clinical hypothyroidism was statistically very significant (P < 0.0001). Copper levels were increasing in clinical hyperthyroidism comparable to euthyroid participants. It was also statistically significant (P < 0.0001). Our study found ceruloplasmin levels in clinical hypothyroidism as 10.44 ± 1.69 ng/mL, Grave's hyperthyroidism 70.85 ± 3.73 ng/mL compared to euthyroid participants 49.17 ± 3.06 ng/mL. Our study observed that both serum copper and ceruloplasmin decreased significantly (P < 0.0001) in hypothyroidism and increase significantly (P < 0.0001) in hyperthyroidism compared to the euthyroid group. This finding was compared with other studies.

Our findings on hypothyroidism were in accordance with studies done by Manisha A et al.,^[10] Pal et al.,^[11] and Alturfan et al.^[12] As per a study conducted by Mittag et al.,^[5] and Valenzise et al.,^[13] serum ceruloplasmin concentration had been reported to be abnormally low in hypothyroid patients. On the other hand, our findings on hyperthyroidism were also in agreement with multiple studies done on the same topic. In a study conducted by Sinha et al.,^[14] both serum copper and ceruloplasmin levels were seen to be significantly high (P < 0.001) in the case group when compared to the control group. A study by Liu et al.^[15] also found that hyperthyroidism was associated with higher copper levels.

The mutual association between thyroid hormones and copper is complex. It can be understood in the following way. Copper is a mineral found the third most in the human body. It plays an essential role in thyroid metabolism, thyroid hormone production, and its absorption. Copper stimulates the creation of thyroxine (T4). It prevents over-absorption of thyroxine in the blood cells by the control calcium levels in the human body. Copper also plays a role in phospholipids synthesis, which stimulates TSH.^[4] Copper acts as a cofactor for the tyrosinase enzyme, which is necessary for tyrosine biosynthesis. Tyrosine is an important component of thyroglobulin, which is needed for thyroid hormone synthesis.^[16] The deficiency of copper can exert both a direct effect on the metabolic process and an indirect effect disturbing iodine metabolism. It decreases protein-bound iodine production by the thyroid gland.^[11]

On the other hand, thyroid hormone levels have a significant effect on serum copper levels. The thyroid hormone synthesizes and exports ceruloplasmin, the hepatic copper-transport protein, into the serum.^[5],[6] Ceruloplasmin is an α2-Globulin containing approximately 95% of the total copper in serum.^[17] Many studies showed ceruloplasmin serum levels to be abnormally low in patients with peripheral resistance to thyroid hormones.^[5],[13] Hypothyroidism impairs gastrointestinal absorption of copper or altered copper distribution.^[11] The thyroid also has an influence on GFR in the kidney in the excretion of this mineral.

Conclusion

In the present study, we concluded that patients with clinical hypothyroidism had statistically significant (P < 0.0001) lower levels of serum copper and ceruloplasmin levels compared to euthyroid controls. The scenario was the opposite in the case of Grave's hyperthyroidism patients. Our study found statistically significant (P < 0.0001) higher levels of serum ceruloplasmin and hence serum copper levels in Grave's hyperthyroidism patients compared to controls. Therefore, patients with hypothyroidism and hyperthyroidism should be routinely screened for serum copper and ceruloplasmin levels for more effective thyroid treatment.

Limitations

The sample size was limited to 60 participants with hyperthyroidism. Hence, the exact relationship is difficult to establish. This study was limited to rural areas around Northern Andhra Pradesh. The exact scenario in India can be understood with widespread research done in all parts of India, including both urban and rural populations.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1.	Unnikrishnan AG, Kalra S, Sahay RK, Bantwal G, John M, Tewari N. Prevalence of hypothyroidism in adults: An epidemiological study in eight cities of India. Indian J Endocrinol Metab 2013;17:647-52.
2.	Unnikrishnan AG, Menon UV. Thyroid disorders in India: An epidemiological perspective. Indian J Endocrinol Metab 2011;15:S78-81.
3.	Burtis CA, Bruns DE. Tietz fundamentals Clinical Chemistry and Molecular Diagnostics. 7^th ed. London: Elsevier Publication; 2014.
4.	Mohammed HI, Idrees OF, Ismail AM, Ahmed SA. Evaluation of copper and zinc among hyper and hypothyroidism patients. Eur Acad Res 2015;3:5422-34.
5.	Mittag J, Behrends T, Nordström K, Anselmo J, Vennström B, Schomburg L. Serum copper as a novel biomarker for resistance to thyroid hormone. Biochem J 2012;443:103-9.
6.	Fitch CA, Song Y, Levenson CW. Developmental regulation of hepatic ceruloplasmin mRNA and serum activity by exogenous thyroxine and dexamethasone. Proc Soc Exp Biol Med 1999;221:27-31.
7.	Diez JJ. Hypothyroidism in patients older than 55 years an analysis of the etiology and assessment of the effectiveness of therapy. J Gerontol Ser A 2002;57:M315-20.
8.	Papadakis MA, McPhee SJ, Rabow MW. Current Medical Diagnosis and Treatment 2013. New York: McGraw-Hill Medical; 2013.
9.	Bagcchi S. Hypothyroidism in India: More to be done. Lancet Diabetes Endocrinol 2014;2:778.
10.	Manisha A, Roshan KM, Sudeep K, Imran M, Sumesh PS. Study of trace elements in patients of hypothyroidism with special reference to zinc and copper. Biomed J Sci Technol Res 2018;6:5190-94. [DOI: 10.26717/ BJSTR.2018.06.001336].
11.	Pal S, Patra S, Mukhopadhyay T. Status of lipid peroxidation and role of serum zinc and copper in subjects with hypothyroidism. IOSR J Dent Med Sci (IOSR-JDMS) 2018;17:54-7.
12.	Alturfan AA, Zengin E, Dariyerli N, Alturfan EE, Gumustas MK, Aytac E, et al. Investigation of zinc and copper levels in methimazole-induced hypothyroidism: Relation with the oxidant-antioxidant status. Folia Biol (Praha) 2007;53:183-8.
13.	Valenzise M, Porcaro F, Zirilli G, De Luca F, Cinquegrani M, Aversa T. Hypoceruloplasminemia: An unusual biochemical finding in a girl with Hashimoto's thyroiditis and severe hypothyroidism. Pediatr Med Chir 2018;40:doi: 10.4081/pmc.2018.179. PMID: 30514077.
14.	Sinha S, Kar K, Dasgupta A, Basu S, Sen S. Correlation of serum zinc with TSH in hyperthyroidism. Asian J Med Sci 2016;7:66-9. [DOI:10.3126/ajms.v7i1.12895].
15.	Liu Y, Liu S, Mao J, Piao S, Qin J, Peng S et al. Serum Trace Elements Profile in Graves' Disease Patients with or without Orbitopathy in Northeast China. Biomed Res Int 2018; 2018:3029379. doi: 10.1155/2018/3029379. PMID: 29546054; PMCID: PMC5818896.
16.	Adedapo KS, Sonuga AA, Afolabi AO, Amosu A. Interaction of some selected trace elements with thyroid hormones in patients with goiter in Ibadan, Nigeria. J Sci Res Rep 2014;3:2875-83.
17.	Bhattacharya A, Deb J, Mondal T, Choudhuri S, Gupta S. Ceruloplasmin and serum MDA levels in hypothyroid patients. Int J Biomed Adv Res 2014;5:369.