https://orcid.org/0000-0002-4708-8420
Abstract
Thyroid hormone analog 3,5,3'-triiodothyroacetic acid (TRIAC) is effective in reducing the hypermetabolism in monocarboxylate transporter 8 (MCT8)–deficient individuals. Because of the structural similarity between TRIAC and 3,3',5'-triiodothyronine (T3), we sought to investigate the degree of cross-reactivity of TRIAC with various commercially available total and free T3 assays.
Varying concentrations (50-1,000 ng/dL) of TRIAC (Sigma Aldrich) were added to pooled serum and assayed for total T3 (TT3) and free T3 (FT3) on the following platforms: e602 (Roche Diagnostics), Architect (Abbott Diagnostics), Centaur (Siemens Healthcare Diagnostics), IMMULITE (Siemens Healthcare Diagnostics), DxI (Beckman Coulter), and Vitros (Ortho Clinical Diagnostics). TT3 competition assay with TRIAC was performed by adding increasing amounts of T3 to pooled serum samples that contained a constant concentration of TRIAC (250 ng/dL).
Significant overestimation of TT3 and FT3 assays were observed across all platforms corresponding to increasing concentrations of TRIAC. The TRIAC effect at 250 ng/dL showed a constant interference of approximately 190 ng/dL TT3.
All commercial TT3 and FT3 assays tested in this work cross-react significantly with TRIAC. Therefore, patients undergoing TRIAC therapy should have T3 hormone response monitored using alternative nonimmunoassay-based methods to avoid misinterpretation of thyroid function profiles.
Evaluate cross-reactivity of thyroid hormone analog 3,5,3'-triiodothyroacetic acid (TRIAC) in hypermetabolism in MCT8-deficient patients in total and free 3,5,3'-triiodothyronine (T3) assays.
TRIAC significantly cross-reacts with various commercially available total and free T3 immunoassays.
Patients undergoing TRIAC therapy should have T3 hormone response monitored by using alternative, nonimmunoassay-based methods to avoid misinterpretation of their thyroid function profile.
Introduction
In 2004, 2 laboratories independently identified patients with alterations in MCT8 gene, also known as SLC16A2, which is located on chromosome X.1,2 Monocarboxylate transporter 8 (MCT8) has been shown to be a specific cell membrane transporter of thyroid hormone.3 The clinical presentation of patients with MCT8 defects is similar, having 2 components: thyroid abnormalities and neuropsychomotor abnormalities.4,5 Characteristic thyroid function test abnormalities are increased serum 3,5,3'-triiodothyronine (T3) and decreased serum 3,3',5'-triiodothyronine, or reverse T3 concentrations. In addition, 3,5,3',5'-tetraiodothyronine (thyroxine, or T4) is reduced in most cases, and thyrotropin (TSH) is normal or slightly elevated. Boys with alterations that produce loss of MCT8 function manifest hypotonia and poor head control in infancy, progressing into spastic quadriplegia and the inability to walk or talk. Although the deficiency of thyroid hormone in the brain causes the neuropsychomotor abnormalities, the high serum T3 level, available to peripheral tissues through alternative transporters, causes hypermetabolism and the inability to gain weight.5
Two analogues of thyroid hormone, 3,5-diiodothyropropionic acid (DITPA) and 3,5,3'-triiodothyroacetic acid (TRIAC), have been found to be effective in reducing hypermetabolism in MCT8-deficient individuals.6,7 Because reduction of T3 is the key to successful treatment, its measurement is of paramount importance for the adjustment of a therapeutic dose. We have previously shown that the Centaur device (Siemens Healthcare Diagnostics) can effectively measure serum T3 with minimal interference from DITPA, which was not significant for the therapeutic serum levels achieved.8 In the current study, we sought to investigate the degree of cross-reactivity of TRIAC with various commercially available routine laboratory measurements of both total T3 (TT3) and free T3 (FT3) on 6 platforms from 5 in vitro diagnostic companies.
Materials and Methods
We prepared 1 mg/mL of TRIAC (Sigma Aldrich) stock solution in ethanol, and then further diluted it 2,000-fold by using phosphate-buffered saline (PBS) to obtain 50 µg/dL of working solution. Varying amounts of TRIAC working solution to a maximum of 105 µL were added to 7 mL final volume of pooled serum (containing approximately a baseline concentration of 42 ng/dL of TT3, as assayed on the e602 platform [Roche Diagnostics]), to create a series with final TRIAC concentrations ranging from 50 to 750 ng/dL. These samples were then assayed for TT3 and FT3 using the following platforms in duplicate: e602, Architect (Abbott Diagnostics), Centaur, and Vitros (Ortho Clinical Diagnostics); samples were performed in singlicate on IMMULITE (Siemens Healthcare Diagnostics) and DxI (Beckman Coulter). Each platform’s analytical performance and assay principles are summarized in Supplemental Table 1 (all supplemental materials can be found at American Journal of Clinical Pathology online). The respective values in the TRIAC-treated samples are shown in Figure 1, with the baseline TT3 subtracted. Measurements of total and free T4 as well as TSH were also carried out. Further investigation was performed to determine if the cross-reactivity by TRIAC could be outcompeted by excess TT3. We added 10 µL of TRIAC working solution to 2mL final volume of pooled serum samples to achieve a final constant concentration of 250 ng/dL; we then added increasing amounts of T3 (10 µg/dL) prepared in a working solution of PBS. These samples were analyzed using the Roche e602 TT3 method in duplicate.
Interference of 3,5,3'-triiodothyroacetic acid (TRIAC) on various total 3,5,3'-triiodothyronine (TT3) (A) and free T3 (FT3) (B) in commercially available assays. Increasing amounts of TRIAC (0-750 ng/dL) were added to a constant baseline serum pool with a baseline TT3 of approximately 42 ng/dL and FT3 of approximately 137 ng/dL as measured on the e602 platform (Roche Diagnostics). Baseline values were subtracted from TT3 and FT3, as determined in samples spiked with TRIAC and measured on various commercial assay platforms. Each sample was assayed in duplicate, with the exception of IMMULITE (Siemens Healthcare Diagnostics) and DxI (Beckman Coulter), which were assayed in singlicate.
Results
In an in vitro spiking experiment performed by adding increasing concentrations of TRIAC to the pooled serum showed that the total and free T4 and TSH measurements were not affected by TRIAC on any of the immunoassay platforms used (Supplemental Table 2). As expected, there was a significant overestimation of TT3 and FT3 assays across all platforms, corresponding to increasing concentrations of TRIAC compared with their baseline, nonspiked samples Figure 1. The degree of TT3 assay susceptibility to TRIAC interference from the highest to the lowest are as follows: IMMULITE > e602 > DxI = Vitros > Architect > Centaur. Similarly, considerable cross-reactivity with TRIAC treatment was observed in the FT3 assays in the following order: Architect > e602 > Centaur. The cross-reactivity of TRIAC in all platforms for TT3 and FT3 assays was dose dependent. When TRIAC was held at a constant concentration, its contribution to the measured TT3 concentration was, on average, 191 ng/dL, or 76% of the concentration of TRIAC present in each sample irrespective of the amount of TT3 added Figure 2.
Effect of total 3,5,3'-triiodothyronine (TT3) on a constant amount of 3,5,3'-triiodothyroacetic acid (TRIAC) on the e602 platform (Roche Diagnostics). Increasing amounts of TT3 were added to respective serum samples with a constant 250 ng/dL TRIAC (squares) or none (circles). Each sample was assayed in duplicate.
Discussion
The structural difference between T3 and its deaminated and decarboxylated analogue, TRIAC, was insufficient to impart specificity of T3 to the monoclonal antibodies used on any platform. This finding contrasts with the greater difference between DITPA, also used in the treatment of MCT8 deficiency, and T3, which lacks iodine in the outer ring as well as an amino group.
Our studies showed that many commercial TT3 and FT3 assays tested in this work cross-react significantly with TRIAC. The dose-dependent nature of the cross-reactivity suggests that antibodies to T3 bind to the same epitope as TRIAC. Therefore, patients undergoing TRIAC therapy should have their T3 hormone response monitored by using alternative assay methodology, such as mass spectrometry, which can distinguish TRIAC from TT3, to avoid misinterpretation of thyroid function profiles.
Funding: This work was supported in part by grant DK15070 from the National Institutes of Health.
Acknowledgments
The authors thank the staff of the Clinical Chemistry Laboratory at the University of Chicago Medical Center for their assistance and support. We also thank Dr Earle W. Holmes from Loyola University Medical Center, who provided assistance with the thyroid function assays on the Centaur analyzer.
References
