Elevated free thyroxine and free triiodothyronine probably caused by high-dose biotin intake in a patient with Graves’ disease: a case report

Abstract

Biotin is a water-soluble vitamin that acts as a cofactor for carboxylase, and is often used as a component in several immunoassays. We present a case of a 46-year-old male with Graves’ disease (GD) who revealed elevated free thyroxine (FT4) and free triiodothyronine (FT3) levels after high-dose biotin intake. Levels of these hormones had been within the reference range when he was on thiamazole 5 mg/day for 7 years; however, the levels increased from 1.04 to 2.20 ng/dL and from 3.05 to 9.84 pg/mL for FT4 and FT3, respectively, after he started taking biotin 72 mg/day. Despite these high levels, his symptoms and the other laboratory results, including the thyroid-stimulating hormone level, did not suggest GD relapse. His thyroid hormone data was decreased and returned within the reference range immediately after the laboratory assays for FT3 and FT4 had been coincidentally changed from those containing streptavidin-biotin complexes to biotin-free ones. Biotin interference, which is caused by high-dose biotin intake and immunoassays using some form of streptavidin-biotin complex, is sometimes clinically problematic, giving high or low results. To our knowledge, this is the first case report of a patient with GD on high-dose biotin receiving high thyroid hormone level results that were initially misunderstood as an aggravation of the disease; there are some reports of misdiagnosis of hyperthyroidism due to biotin administration. Unexpected fluctuations in thyroid function test results in patients with GD should be checked for biotin intake, immunoassays and the limiting concentration of biotin to avoid misdiagnosis of relapse.

GRAVES’ DISEASE (GD) is an organ-specific autoimmune disease that causes hyperthyroidism due to circulating autoantibodies that activate thyroid-stimulating hormone (TSH) receptors [1]. Etiology of GD lies in genetic factors [2, 3]; however, environmental factors, such as smoking, high iodine intake, stress, and pregnancy, can also predispose individuals to GD [4-6]. Relapse of GD is common, especially during antithyroid drug (ATD) therapy [7]. Predictors of relapse in GD include orbitopathy, smoking, thyroid volume measured by sonography, goiter size, and levels of free thyroxine (FT4), total triiodothyronine, anti-TSH receptor antibody, and thyrotropin binding inhibiting immunoglobulin (TBII) [8]. According to a study on the long-term prognosis of 549 patients with Graves’ hyperthyroidism initially treated with thionamides, a longer TBII normalization time, and a higher final thyroid weight were associated with a poor prognosis, and remission mostly occurred after 4–11 years of treatment [9]. There are no reliable studies on the frequency of GD relapse during the titration of ATDs; however, it is commonly noticed.

Electrochemiluminescence immunoassays (ECLIA) are widely preferred in thyroid function tests over radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA) for several reasons, including enhanced detection limits and economic efficiency [10, 11]. Some substances have been reported to interfere with free thyroid hormone assay, including biotin, anti-streptavidin antibodies, heterophilic antibodies, anti-thyroid autoantibodies, and anti-Ru antibodies [12-14]. Biotin is a water-soluble vitamin that acts as a cofactor for carboxylase [15] and is commonly used as a supplement to promote the health of skin and hair [16]. High doses of biotin can disturb laboratory data, because the interaction between biotin and streptavidin is used in various immunoassays. This phenomenon is known as “biotin interference” [16-19]. We report a case in which biotin interference led to a misunderstanding of GD relapse.

Case Report

A 46-year-old man was diagnosed with GD and started taking thiamazole (MMI) 11 years prior. He had experienced multiple relapses of GD, but had chosen to continue treatment with oral medication, rather than selecting radioactive iodine treatment or surgery. He was taking MMI 5 mg/day for 7 years. The clinical course of the patient is shown in Fig. 1. One year prior to his visit to our department (day –408), his thyroid function test results were as follows: FT4, 1.04 ng/dL (reference range: 0.71–1.12 ng/dL); free triiodothyronine (FT3), 3.05 pg/mL (reference range: 2.49–3.58 pg/dL), TSH, 2.888 μIU/mL (reference range: 0.453–4.219 μIU/mL), and thyrotropin receptor antibody (TRAb) <0.8 IU/L (reference range: <2.0). At that time, he thought he might have had biotin deficiency, based on his history of palmoplantar pustulosis; therefore, he was prescribed biotin 72 mg/day at another clinic (We contacted the clinic’s physician; it was unclear why he had chosen that specific dose). He was taking this high-dose biotin for 11 months prior to his present visit. His FT4 level had increased to 1.56 ng/dL on day –219, and his FT4 and FT3 levels on day 0 were further increased to 2.20 ng/dL and 9.84 pg/mL, respectively. In contrast, the TSH level on day 0 was 3.923 μIU/mL, which was within the reference range. He had no other signs of GD relapse; his body weight was 60 kg, with no recent significant weight loss. He was conscious, with a blood pressure of 120/68 mmHg and a pulse rate of 64 beats per minute, and no recent markedly elevated blood pressure or tachycardia. He did not have hand tremors or increased sweating. Other laboratory data did not reveal any findings that would suggest GD relapse, such as elevated transaminase or alkaline phosphatase (Table 1). MMI was increased to 10 mg/day due to suspicion of disease aggravation, as the patient had experienced relapses in the past. After a discussion with the endocrinology department of the hospital, we strongly suspected that the thyroid hormone data might have been false due to the high-dose biotin administration. From day 26 (days 26, 61, and 152), the patient was instructed to continuously withdraw biotin intake for 48 h before testing. The results of the retest indicated that FT3 and FT4 were within the reference range (reference range of the new test method: FT3 2.52–4.06 pg/mL, FT4 0.75–1.45 ng/dL). However, TSH rose to 5.042 μIU/mL (new reference range: 0.61–4.23 μIU/mL) after the increment in MMI dosage; therefore, the dosage of the drug was reduced again to 5 mg/day. Coincidentally, the thyroid hormone measurement method was changed in the same month, and an assay without biotin was adopted (Previous reagents: Access Free T3, Access Free T4 and Access TSH 3rd IS, Previous analyzer: Access2 PRO, all manufactured by Beckman Coulter; New reagents: Lumipulse Presto FT3, Lumipulse Presto FT4 and Lumipulse Presto TSH IFCC, New analyzer: Accuraseed, all manufactured by Fujirebio Inc.) Thereafter, both FT3 and FT4 values remained within the reference range under MMI 5 mg/day (Fig. 1). We clinically suspected that these were elevated values because of biotin involvement during the course; however, our suspicion could not be ascertained because we did not store past samples and retests with the new immunoassay.

Fig. 1

The clinical course of the patient

The dashed line represents the reference range. FT3: free triiodothyronine, FT4: free thyroxine, MMI: Thiamazole, TSH: thyroid-stimulating hormone

Table 1 Laboratory findings on day 0

Complete blood count		(reference range)	Biochemistry		(reference range)
WBC	6,200/μL	3,300–8,600	AST	18 U/L	13–30
RBC	5.24 × 106/μL	4.35–5.55	ALT	19 U/L	10–42
Hb	15.7 g/dL	13.7–16.8	GGT	15 U/L	13–64
Ht	44.4%	40.7–50.1	ALP	251 U/L	106–322
PLT	246 × 103/μL	158–348	UN	15.8 mg/dL	8.0–20.0
Hormones		(reference range)	Cre	0.76 mg/dL	0.65–1.07
FT3	9.84 pg/mL	2.49–3.58	eGFR	87 mL/min/1.73 m2	60≤
FT4	2.2 ng/dL	0.71–1.12	T-Cho	167 mg/dL	142–248
TSH	3.923 μIU/mL	0.453–4.219	HDL-C	65 mg/dL	38–90
			TG	51 mg/dL	<150
			nonHDL-C	102 mg/dL	<170
			LDL-C	92 mg/dL	70–139

WBC: white blood cell, RBC: red blood cell, Hb: hemoglobin, Ht: hematocrit, PLT: platelet, FT3: free triiodothyronine, FT4: free thyroxine, TSH: thyroid stimulating hormone, AST: aspartate aminotransferase, ALT: alanine aminotransferase, GGT: γ-glutamyl transpeptidase, ALP: alkaline phosphatase, UN: urea nitrogen, Cre: creatinin, eGFR: estimated glomerular filtration rate, T-Cho: total cholesterol, HDL-C: high-density lipoprotein cholesterol, TG: triglyceride, nonHDL-C: non-high-density lipoprotein cholesterol, LDL-C: low-density lipoprotein cholesterol

Discussion

GD is an autoimmune disease that causes hyperthyroidism due to autoantibodies that stimulate TSH receptors [1]. Relapse of GD is common, especially with ATD therapy [7].

Biotin is a water-soluble vitamin that acts as a cofactor for carboxylase, an important enzyme involved in the metabolism of fatty acids, glucose, and amino acids. Biotin deficiency can cause neurological disorders and skin abnormalities [15]. Biotin plays an important role in the treatment of several inherited metabolic disorders, and is also used as supportive therapy for patients with mitochondrial energy metabolism disorders, such as biotin-thiamine-responsive basal ganglia disease and biotinidase deficiency [20]. There are also dietary supplements available, which contain up to 100 times the recommended daily biotin intake (30 μg) or even 30 mg that are used as an effective supplement for the skin and hair [16, 19].

The streptavidin-biotin complex is used in several immunoassays, and it is often clinically problematic that high doses of biotin can cause false results with such assays. This phenomenon is known as biotin interference [16-19]. These immunoassays could produce either a high or low results due to biotin intake, depending on the assay methods. In the competitive assay, biotin in the blood competes with exogenously added biotinylated antigen for binding to streptavidin-coated magnetic particles, resulting in a decreased signal and a high value. In contrast, in the sandwich assay, biotin in blood competes with the biotinylated sandwich complex for binding to streptavidin-coated magnetic particles, resulting in a low value [18]. Nevertheless, biotin-based immunoassays are widely used for this purpose. For example, >50% of the thyroid function testing assays available in France in 2017 utilized biotin-streptavidin complexes [21]. Of the 374 methods performed on the eight most common immunoassay analyzers used in the United States in 2016, biotin was used in 221 [18].

The mechanism of the high result caused by biotin in the competitive assay is as follows: Take FT3 for example, FT3 in the patient’s serum competes with biotin-labeled diiodothyronine in the reagent to bind to alkaline phosphatase-labeled anti-T3 monoclonal antibody, and biotin-labeled diiodothyronine forms a complex by binding to streptavidin-bound magnetic beads. Since only the amount bound to the magnetic beads emit luminescence, the amount of luminescence decreases with an elevation in serum FT3 and increases with a drop in serum FT3. When the biotin concentration in the blood increases due to high biotin intake, the biotin in the serum competitively binds to the streptavidin-bound magnetic beads, resulting in a decrease in luminescence, even though the amount of FT3 in the serum is not actually high. This results in a high reading of the FT3 level. In the present case, both FT3 and FT4 reagents (Access Free T3 and Access Free T4, both manufactured by Beckman Coulter) were previously used in a competitive assay with a streptavidin-biotin complex, and it is considered that the increase in the blood biotin concentration due to biotin intake caused the elevation of FT4 and FT3. According to Beckman Coulter, the limit concentration of biotin interference was 10 ng/mL for both FT3 and FT4 kits. In this case, the test results of FT3 and FT4 were highly variable from day –321 to day 0, although the daily dose of biotin remained unchanged. This result was probably influenced by different serum biotin concentrations, due to differences in the interval between the time of daily biotin intake and the time of blood sampling. In fact, on day –86 (FT3 3.86 pg/mL, FT4 1.20 ng/dL), the time of blood sampling was 13:11, and on day 0 (FT3 9.84 pg/mL, FT4 2.20 ng/dL), it was 9:37. The TSH assays, both the former (Access TSH 3rd IS) and the new (Lumipulse Presto TSH IFCC), were biotin-free and thus completely unaffected by biotin during the course of the case. The patient was subjected to biotin withdrawal for 48 h before blood sampling for the retest; coincidentally, both the FT3 and FT4 assays used within the department had also been changed to biotin-free ones at the same time. Thereafter, the FT3 and FT4 levels remained within the reference range.

In this case, the elevation of free thyroid hormone levels caused by biotin interference was mistaken for an aggravation of GD owing to the high biotin intake. (It was not accompanied by a decrease in TSH; therefore, it was necessary to consider diseases other than GD relapse, such as the syndrome of inappropriate TSH secretion.) Although numerous cases of biotin administration causing misdiagnosis of GD have been reported [16, 22-29], to our knowledge, this is the first case report in which a patient with GD was prescribed high-dose biotin and misdiagnosed as having a disease relapse. When a significant change in thyroid function test results is observed, which is inconsistent with the clinical course, symptoms, or other laboratory findings, it should be confirmed whether the patient is taking biotin, whether the immune assay used can be subject to biotin interference, and if so, the limiting concentration of biotin. Furthermore, if the patient is taking biotin, the type of assay used to determine the thyroid hormone levels and/or biotin withdrawal should be considered.

Ethics Statement

The patient had provided a written informed consent for the publication of this study and its accompanying images. The identity of the patient has been protected.

Acknowledgements

We would like to thank Editage (www.editage.com) for English language editing.

Disclosure

Nozomu Kamei received honoraria from Eli Lilly Japan and Sumitomo Pharma. The other authors declare no conflicts of interest.

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