Agreement of Immunoassay and Tandem Mass Spectrometry in the Analysis of Cortisol and Free T4: Interpretation and Implications for Clinicians

Objective. To quantify differences in results obtained by immunoassays (IAs) and tandem mass spectrometry (MSMS) for cortisol and free thyroxine (FT4). Design & Patients. Cortisol was measured over 60 minutes following a standard ACTH stimulation test (n = 80); FT4 was measured over time in two cohorts of pregnant (n = 57), and nonpregnant (n = 28) women. Measurements. Samples were analyzed with both IA and MSMS. Results. Results for cortisol by the two methods tended to agree, but agreement weakened over the 60-minute test and was worse for higher (more extreme) concentrations. The results for FT4 depended on the method. IA measurements tended to agree with MSMS measurements when values fell within “normal levels”, but agreement was not constant across trimester in pregnant women and was poorest for the extreme (low/high) concentrations. Correlations between MSMS measurements and the difference between MSMS and IA results were strong and positive (0.411 < r < 0.823; all P < .05). Conclusions. IA and MSMS provide different measures of cortisol and FT4 at extreme levels, where clinical decision making requires the greatest precision. Agreement between the methods is inconsistent over time, is nonlinear, and varies with the analyte and concentrations. IA-based measurements may lead to erroneous clinical decisions

Short-Term Time Trends in Prescribing Therapy for Hypothyroidism: Results of a Survey of American Thyroid Association Members

Objective: Hypothyroid patients frequently request specific therapies from their physicians. Combination therapy is vigorously discussed at professional meetings. We wished to determine if physician prescribing patterns for hypothyroidism changed during 2017 after specific educational events.Methods: A survey addressing treatment of hypothyroidism was emailed to American Thyroid Association (ATA) members on three occasions in 2017. The Spring emails were sent prior to a satellite symposium addressing hypothyroidism, and prior to the annual Endocrine Society and ATA meetings; the December emails were sent after these events. Physicians were presented with thirteen theoretical patients and chose from 6 therapeutic options, including levothyroxine, synthetic combination therapy, thyroid extract, and liothyronine monotherapy. The patient scenarios successively incorporated factors potentially providing reasons for considering combination therapy. Multivariate repeated measures logistic regression analyses first examined effects of physician characteristics on prescribing the various therapies. Then, analyses also incorporated timing, by comparing prescribing patterns in February, March, and December.Results: In analyses of prescribing levothyroxine monotherapy vs. any T3 therapy, there was a trend of borderline significance (p = 0.053) for T3 therapy to be prescribed more in December compared with February-March combined. When multivariate analyses were performed controlling for time and physician characteristics, choice of therapy was only significantly affected by country of practice (OR 1.7, CI 1.3–2.2). Physician choice of therapies was also examined for the options of continuing (1) levothyroxine, vs. (2) increasing levothyroxine, (3) adding liothyronine either with or without levothyroxine reduction, or (4) replacing levothyroxine with desiccated thyroid extract or liothyronine. When multivariate analyses incorporating time and physician characteristics were performed, respondents in December (OR 1.5, CI 1.0–2.3) and those practicing in North America (OR 1.8, CI 1.2–2.6) were more likely to prescribe liothyronine.Conclusions: This survey shows that although current North American guidelines do not recommend combination therapy, such therapy is being prescribed more over time and is also more commonly prescribed in North America. It is possible our guidelines are failing to incorporate evidence that physicians are considering when prescribing combination therapy. Such evidence could include data about patient preferences, and this needs to be a focus of future studies

3,5-T2—A Janus-Faced Thyroid Hormone Metabolite Exerts Both Canonical T3-Mimetic Endocrine and Intracrine Hepatic Action

Over the last decades, thyroid hormone metabolites (THMs) received marked attention as it has been demonstrated that they are bioactive compounds. Their concentrations were determined by immunoassay or mass-spectrometry methods. Among those metabolites, 3,5-diiodothyronine (3,5-T2), occurs at low nanomolar concentrations in human serum, but might reach tissue concentrations similar to those of T4 and T3, at least based on data from rodent models. However, the immunoassay-based measurements in human sera revealed remarkable variations depending on antibodies used in the assays and thus need to be interpreted with caution. In clinical experimental approaches in euthyroid volunteers and hypothyroid patients using the immunoassay as the analytical tool no evidence of formation of 3,5-T2 from its putative precursors T4 or T3 was found, nor was any support found for the assumption that 3,5-T2 might represent a direct precursor for serum 3-T1-AM generated by combined deiodination and decarboxylation from 3,5-T2, as previously documented for mouse intestinal mucosa. We hypothesized that lowered endogenous production of 3,5-T2 in patients requiring T4 replacement therapy after thyroidectomy or for treatment of autoimmune thyroid disease, compared to production of 3,5-T2 in individuals with intact thyroid glands might contribute to the discontent seen in a subset of patients with this therapeutic regimen. So far, our observations do not support this assumption. However, the unexpected association between high serum 3,5-T2 and elevated urinary concentrations of metabolites related to coffee consumption requires further studies for an explanation. Elevated 3,5-T2 serum concentrations were found in several situations including impaired renal function, chronic dialysis, sepsis, non-survival in the ICU as well as post-operative atrial fibrillation (POAF) in studies using a monoclonal antibody-based chemoluminescence immunoassay. Pilot analysis of human sera using LC-linear-ion-trap-mass-spectrometry yielded 3,5-T2 concentrations below the limit of quantification in the majority of cases, thus the divergent results of both methods need to be reconciliated by further studies. Although positive anti-steatotic effects have been observed in rodent models, use of 3,5-T2 as a muscle anabolic, slimming or fitness drug, easily obtained without medical prescription, must be advised against, considering its potency in suppressing the HPT axis and causing adverse cardiac side effects. 3,5-T2 escapes regular detection by commercially available clinical routine assays used for thyroid function tests, which may be seriously disrupted in individuals self-administering 3,5-T2 obtained over-the counter or from other sources

3,5-T2-A Janus-Faced Thyroid Hormone Metabolite Exerts Both Canonical T3-Mimetic Endocrine and Intracrine Hepatic Action.

Over the last decades, thyroid hormone metabolites (THMs) received marked attention as it has been demonstrated that they are bioactive compounds. Their concentrations were determined by immunoassay or mass-spectrometry methods. Among those metabolites, 3,5-diiodothyronine (3,5-T2), occurs at low nanomolar concentrations in human serum, but might reach tissue concentrations similar to those of T4 and T3, at least based on data from rodent models. However, the immunoassay-based measurements in human sera revealed remarkable variations depending on antibodies used in the assays and thus need to be interpreted with caution. In clinical experimental approaches in euthyroid volunteers and hypothyroid patients using the immunoassay as the analytical tool no evidence of formation of 3,5-T2 from its putative precursors T4 or T3 was found, nor was any support found for the assumption that 3,5-T2 might represent a direct precursor for serum 3-T1-AM generated by combined deiodination and decarboxylation from 3,5-T2, as previously documented for mouse intestinal mucosa. We hypothesized that lowered endogenous production of 3,5-T2 in patients requiring T4 replacement therapy after thyroidectomy or for treatment of autoimmune thyroid disease, compared to production of 3,5-T2 in individuals with intact thyroid glands might contribute to the discontent seen in a subset of patients with this therapeutic regimen. So far, our observations do not support this assumption. However, the unexpected association between high serum 3,5-T2 and elevated urinary concentrations of metabolites related to coffee consumption requires further studies for an explanation. Elevated 3,5-T2 serum concentrations were found in several situations including impaired renal function, chronic dialysis, sepsis, non-survival in the ICU as well as post-operative atrial fibrillation (POAF) in studies using a monoclonal antibody-based chemoluminescence immunoassay. Pilot analysis of human sera using LC-linear-ion-trap-mass-spectrometry yielded 3,5-T2 concentrations below the limit of quantification in the majority of cases, thus the divergent results of both methods need to be reconciliated by further studies. Although positive anti-steatotic effects have been observed in rodent models, use of 3,5-T2 as a muscle anabolic, slimming or fitness drug, easily obtained without medical prescription, must be advised against, considering its potency in suppressing the HPT axis and causing adverse cardiac side effects. 3,5-T2 escapes regular detection by commercially available clinical routine assays used for thyroid function tests, which may be seriously disrupted in individuals self-administering 3,5-T2 obtained over-the counter or from other sources

3,5-T2-A Janus-Faced Thyroid Hormone Metabolite Exerts Both Canonical T3-Mimetic Endocrine and Intracrine Hepatic Action.

Over the last decades, thyroid hormone metabolites (THMs) received marked attention as it has been demonstrated that they are bioactive compounds. Their concentrations were determined by immunoassay or mass-spectrometry methods. Among those metabolites, 3,5-diiodothyronine (3,5-T2), occurs at low nanomolar concentrations in human serum, but might reach tissue concentrations similar to those of T4 and T3, at least based on data from rodent models. However, the immunoassay-based measurements in human sera revealed remarkable variations depending on antibodies used in the assays and thus need to be interpreted with caution. In clinical experimental approaches in euthyroid volunteers and hypothyroid patients using the immunoassay as the analytical tool no evidence of formation of 3,5-T2 from its putative precursors T4 or T3 was found, nor was any support found for the assumption that 3,5-T2 might represent a direct precursor for serum 3-T1-AM generated by combined deiodination and decarboxylation from 3,5-T2, as previously documented for mouse intestinal mucosa. We hypothesized that lowered endogenous production of 3,5-T2 in patients requiring T4 replacement therapy after thyroidectomy or for treatment of autoimmune thyroid disease, compared to production of 3,5-T2 in individuals with intact thyroid glands might contribute to the discontent seen in a subset of patients with this therapeutic regimen. So far, our observations do not support this assumption. However, the unexpected association between high serum 3,5-T2 and elevated urinary concentrations of metabolites related to coffee consumption requires further studies for an explanation. Elevated 3,5-T2 serum concentrations were found in several situations including impaired renal function, chronic dialysis, sepsis, non-survival in the ICU as well as post-operative atrial fibrillation (POAF) in studies using a monoclonal antibody-based chemoluminescence immunoassay. Pilot analysis of human sera using LC-linear-ion-trap-mass-spectrometry yielded 3,5-T2 concentrations below the limit of quantification in the majority of cases, thus the divergent results of both methods need to be reconciliated by further studies. Although positive anti-steatotic effects have been observed in rodent models, use of 3,5-T2 as a muscle anabolic, slimming or fitness drug, easily obtained without medical prescription, must be advised against, considering its potency in suppressing the HPT axis and causing adverse cardiac side effects. 3,5-T2 escapes regular detection by commercially available clinical routine assays used for thyroid function tests, which may be seriously disrupted in individuals self-administering 3,5-T2 obtained over-the counter or from other sources

Agreement of Immunoassay and Tandem Mass Spectrometry in the Analysis of Cortisol and Free T4: Interpretation and Implications for Clinicians

Objective. To quantify differences in results obtained by immunoassays (IAs) and tandem mass spectrometry (MSMS) for cortisol and free thyroxine (FT4). Design &amp; Patients. Cortisol was measured over 60 minutes following a standard ACTH stimulation test (n = 80); FT4 was measured over time in two cohorts of pregnant (n = 57), and nonpregnant (n = 28) women. Measurements. Samples were analyzed with both IA and MSMS. Results. Results for cortisol by the two methods tended to agree, but agreement weakened over the 60-minute test and was worse for higher (more extreme) concentrations. The results for FT4 depended on the method. IA measurements tended to agree with MSMS measurements when values fell within &quot;normal levels&quot;, but agreement was not constant across trimester in pregnant women and was poorest for the extreme (low/high) concentrations. Correlations between MSMS measurements and the difference between MSMS and IA results were strong and positive (0.411 &lt; r &lt; 0.823; all P &lt; .05). Conclusions. IA and MSMS provide different measures of cortisol and FT4 at extreme levels, where clinical decision making requires the greatest precision. Agreement between the methods is inconsistent over time, is nonlinear, and varies with the analyte and concentrations. IA-based measurements may lead to erroneous clinical decisions

Demonstration of reciprocal diurnal variation in human serum T3 and rT3 concentration demonstrated by mass spectrometric analysis and establishment of thyroid hormone reference intervals.

Background: There has been a wide range of reference intervals proposed in previous literature for thyroid hormones due to large between-assay variability of immunoassays, as well as lack of correction for collection time. We provided the diurnal reference intervals for five thyroid hormones, namely total thyroxine (TT4), total triiodothyronine (TT3), free thyroxine (FT4), free triiodothyronine (FT3), and reverse T3 (rT3), measured in serum samples of healthy participants using a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method. Methods: Couplet serum samples (a.m. and p.m.) were collected from 110 healthy females and 49 healthy males. Healthy volunteers were recruited from four participating centers between 2016 and 2018. Measurements of thyroid hormones were obtained by LC-MS/MS analysis. Results: Our study revealed significant uptrend in AM to PM FT4 ( Conclusion: When diagnosing thyroid disorders, it is important to have accurate measurement of thyroid hormones, and to acknowledge the diurnal fluctuation found, especially for FT3. Our study highlights the importance of standardization of collection times and implementation of LC-MS/MS in thyroid hormone measurement

Prognosis of Differentiated Thyroid Cancer in Relation to Serum Thyrotropin and Thyroglobulin Antibody Status at Time of Diagnosis

BACKGROUND: Serum thyrotropin (TSH) concentration and thyroid autoimmunity may be of prognostic importance in differentiated thyroid cancer (DTC). Preoperative serum TSH level has been associated with higher DTC stage in cross-sectional studies; data are contradictory on the significance of thyroid autoimmunity at the time of diagnosis. OBJECTIVE: We sought to assess whether preoperative serum TSH and perioperative antithyroglobulin antibodies (TgAb) were associated with thyroid cancer stage and outcome in DTC patients followed by the National Thyroid Cancer Treatment Cooperative Study, a large multicenter thyroid cancer registry. METHODS: Patients registered after 1996 with available preoperative serum TSH (n=617; the TSH cohort) or perioperative TgAb status (n=1770; the TgAb cohort) were analyzed for tumor stage, persistent disease, recurrence, and overall survival (OS; median follow-up, 5.5 years). Parametric tests assessed log-transformed TSH, and categorical variables were tested with chi square. Disease-free survival (DFS) and OS was assessed with Cox models. RESULTS: Geometric mean serum TSH levels were higher in patients with higher-stage disease (Stage III/IV=1.48 vs. 1.02 mU/L for Stages I/II; p=0.006). The relationship persisted in those aged ≥45 years after adjusting for sex (p=0.01). Gross extrathyroidal extension (p=0.03) and presence of cervical lymph node metastases (p=0.003) were also significantly associated with higher serum TSH. Disease recurrence and all-cause mortality occurred in 37 and 38 TSH cohort patients respectively, which limited the power for survival analysis. Positive TgAb was associated with lower stage on univariate analysis (positive TgAb in 23.4% vs. 17.8% of Stage I/II vs. III/IV patients, respectively; p=0.01), although the relationship lost significance when adjusting for age and sex (p=0.34). Perioperative TgAb was not an independent predictor of DFS (hazard ratio=1.12 [95% confidence interval=0.74-1.69]) or OS (hazard ratio=0.98 [95% confidence interval=0.56-1.72]). CONCLUSIONS: Preoperative serum TSH level is associated with higher DTC stage, gross extrathyroidal extension, and neck node metastases. Perioperative TgAb is not an independent predictor of DTC prognosis. A larger cohort is required to assess whether preoperative serum TSH level predicts recurrence or mortality

An online survey of hypothyroid patients demonstrates prominent dissatisfaction

Background: Approximately 15% more patients taking levothyroxine (LT4) report impaired quality of life compared to controls. This could be explained by additional diagnoses independently affecting quality of life and complicating assignment of causation. This study sought to investigate the underpinnings of reduced quality of life in hypothyroid patients and to provide data for discussion at a symposium addressing hypothyroidism. Methods: An online survey for hypothyroid patients was posted on the American Thyroid Association Web site and forwarded to multiple groups. Respondents were asked to rank satisfaction with their treatment for hypothyroidism and their treating physician. They also ranked their perception regarding physician knowledge about hypothyroidism treatments, need for new treatments, and life impact of hypothyroidism on a scale of 1–10. Respondents reported the therapy they were taking, categorized as LT4, LT4 and liothyronine (LT4 + LT3), or desiccated thyroid extract (DTE). They also reported sex, age, cause of hypothyroidism, duration of treatment, additional diagnoses, and prevalence of symptoms. Results: A total of 12,146 individuals completed the survey. The overall degree of satisfaction was 5 (interquartile range [IQR] = 3–8). Among respondents without self-reported depression, stressors, or medical conditions (n = 3670), individuals taking DTE reported a higher median treatment satisfaction of 7 (IQR = 5–9) compared to other treatments. At the same time, the LT4 treatment group exhibited the lowest satisfaction of 5 (IQR = 3–7), and for the LT4 + LT3 treatment group, satisfaction was 6 (IQR = 3–8). Respondents taking DTE were also less likely to report problems with weight management, fatigue/energy levels, mood, and memory compared to those taking LT4 or LT4 + LT3. Conclusions: A subset of patients with hypothyroidism are not satisfied with their current therapy or their physicians. Higher satisfaction with both treatment and physicians is reported by those patients on DTE. While the study design does not provide a mechanistic explanation for this observation, future studies should investigate whether preference for DTE is related to triiodothyronine levels or other unidentified causes