Levothyroxine is a commonly prescribed medication for hypothyroidism, and many cases of acute ingestion of excess levothyroxine have been reported, such as young children ingesting tablets by accident [3], adults attempting to commit suicide [4] [5] [6] [7]. Edmundo K et al. described an adult case of accidental intoxication with 50 mg/day instead of 50 μg/day of levothyroxine over 9 days, due to pharmacist error in the preparation of the capsules. The patient presented with a stuporous mental state, atrial fibrillation and acute respiratory failure, but treatment with charcoal hemoperfusion was successful [8]. In most of the cases mentioned above, the patients was not in critical condition in spite of the excessive intake of levothyroxine, ranging from 2 mg to 720 mg. FT4 levels in those cases ranged from 38.7 pmol/L to >167 pmol/L [3] [4] [5] [6] [7]. These reports suggested that clinical course severity did not always depend on the amount of levothyroxine ingested or on the patient’s free thyroid hormone level, though pre-hospital deaths were not included.
In terms of thyroid hormone action, more than 99% of T4 and T3 are pooled in serum in binding forms with thyroxine binding globulin, prealbumin and albumin [9]. Thyroid hormone activity is produced by FT3. Serum FT3 enters cytoplasm through thyroid hormone transporters, specific transporters for T3 or T4. Next, T3 is pooled in cytoplasm following conversion of T4 to T3 by iodothyronine deiodinase [10]. Finally, T3 binds to nuclear T3 receptors to initiate transactivation. Thus, there are various binding sites in serum and cytoplasm which are capable of storing T3 and T4. In the present case, the patient had a benign clinical course despite temporal toxic exposure to levothyroxine. As she had thyroid dyshormonogenesis, it was indicated that the prior discontinuation of levothyroxine for a month caused severe hypothyroidism before massive ingestion, which increased the potential vacant binding sites of thyroid hormone binding proteins as a buffer to prevent toxic T3 effect.
Ishihara T et al. reported an athyreotic patient having taken 2 mg of levothyroxine at one time, with serum concentrations of T4, FT4 and reverse T3 (rT3) that reached a peak on the second day, while the serum T3 level peaked one day later [7]. FT3 level in the present case also peaked later, indicating that the toxic dose of levothyroxine partly changed to T3 during the two days. Ishihara T et al. also pointed out that the maximum concentrations of T4, FT4 and rT3 were very high, while the peak T3 level did not exceed the upper limit of the normal range. No signs or symptoms of thyroid toxicity other than mild tachycardia were noted. The patient exhibited a low T3/T4 ratio, and it was speculated in that report that the thyroid type 1 deiodinase activity in the thyroid was one of the major determinants in the metabolic clearance of serum T4. Furthermore, there was a report that the mean FT3/FT4 ratios in Graves’ disease and destruction-induced thyrotoxicosis were 0.395 and 0.287, respectively [11]. Compared to that study, the FT3/FT4 ratios in the present case, 0.11 on the first day and 0.15 on the third day (Supplementary Table 1), were obviously low. Another group reported that the FT3/FT4 ratios after total thyroidectomy were lower than those before the surgery in patients with papillary thyroid carcinoma, demonstrating that the median ratio was 0.31 (0.28–0.34; 25th to 75th percentiles) on pre-thyroidectomy and 0.25 (0.22–0.28) on post-thyroidectomy [12]. It is known that almost 80% of T3 is derived peripherally from T4, and about 20% of T3 is secreted directly from the thyroid in healthy subjects. The half-life of T3 is about one day, which is much shorter than that of T4, which is about seven days. Therefore, it is considered that T3 level in thyroid dyshormonogenesis could be increased remarkably only if there is a continuous, sufficient supply of T4. All T3 in our patient was considered to be derived from T4; thus, her FT3 level, which represented thyroid hormone activity, would had been lower than that of people with normal thyroid function in the same situation. Thus, it was reasonably speculated that the lack of intra-thyroidal T3 production caused by iodide organification defect could protect her against the overdose of levothyroxine.
Concerning methodology for measurement of thyroid hormone, various immunoassays are commercially available. Thirteen in vitro diagnostic manufacturers participated in the Phase IV methods comparison study of standardization of free thyroxine measurements, and the upper limits of measurement intervals for FT4 by those immunoassays were listed as 77–155 pmol/L [13]. Not only by levothyroxine overdose, but also in daily medical practice, we sometimes see a patient, typically one with Grave’s disease, whose FT4 or FT3 levels exceed the upper measurement limit. It is generally impossible by immunoassay to accurately determine real concentration when it is over the measurement range. Using LC-MS/MS, however, higher concentrations are able to be determined. As a result of measuring FT4 by ultrafiltration LC-MS/MS in the present case, it was revealed that FT4 level was tremendously increased compared to FT3 level on the first day, and was decreased on the third day. It should be noted that ultrafiltration is distinct from the standard method, which is equilibrium dialysis. Nevertheless, a previous comparison study evaluated an ultrafiltration plus LC-MS/MS assay for FT4, and reported that the correlation between the ultrafiltration LC-MS/MS and the gold standard equilibrium dialysis methods was excellent (r = 0.954) [14].
In the present case, the FT3 levels measured using ultrafiltration LC-MS/MS were differed from those using ECLIA. Currently, FT4 and FT3 measurements have not been standardized. The International Federation of Clinical Chemistry Committee for Standardization of Thyroid Function Tests compared the results for measurement of a panel of single donor sera using clinical laboratory procedures (17 for FT4, 14 for FT3) based on equilibrium dialysis-isotope dilution-mass spectrometry and immunoassays [15]. With regard to FT3, they reported that the biases were distributed from -30% to +22%. Therefore, free thyroid hormone levels would be disparate between LC-MS/MS and immunoassays including ECLIA.
In conclusion, the serum level of FT4 after the ingestion of 5 mg of levothyroxine in the present case with hypothyroidism increased above the measurable range of immunoassay. More specific FT4 concentrations were determined using ultrafiltration LC-MS/MS, which revealed that the value of FT4 was extremely high compared to that of FT3. These results indicate that ultrafiltration LC-MS/MS is worth utilizing for measuring free thyroid hormone levels in thyrotoxic cases when the level is over immunoassay’s upper measurement limit. Though LC-MS/MS is currently a time-consuming technique, it is expected to be developed into a routine method in clinical laboratories in the near future, which will lead to more appropriate management of extreme thyrotoxicosis.