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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., (Copyright © 2020 Köhrle, Lehmphul, Pietzner, Renko, Rijntjes, Richards, Anselmo, Danielsen and Jonklaas.)

Background: In numerous studies based predominantly on rodent models, administration of 3,5-diiodo-L-thyronine (3,5-T2), a metabolite of the thyroid hormones (TH) thyroxine (T4) and triiodo-L-thyronine (T3), was reported to cause beneficial health effects, including reversal of steatohepatosis and prevention of insulin resistance, in most instances without adverse thyrotoxic side effects. However, the empirical evidence concerning the physiological relevance of endogenously produced 3,5-T2 in humans is comparatively poor. Therefore, to improve the understanding of 3,5-T2-related metabolic processes, we performed a comprehensive metabolomic study relating serum 3,5-T2 concentrations to plasma and urine metabolite levels within a large general population sample. Methods: Serum 3,5-T2 concentrations were determined for 856 participants of the population-based Study of Health in Pomerania-TREND (SHIP-TREND). Plasma and urine metabolome data were generated using mass spectrometry and nuclear magnetic resonance spectroscopy, allowing quantification of 613 and 578 metabolites in plasma and urine, respectively. To detect thyroid function-independent significant 3,5-T2-metabolite associations, linear regression analyses controlling for major confounders, including thyrotropin and free T4, were performed. The same analyses were carried out using a sample of 16 male healthy volunteers treated for 8 weeks with 250 μg/day levothyroxine to induce thyrotoxicosis. Results: The specific molecular fingerprint of 3,5-T2 comprised 15 and 73 significantly associated metabolites in plasma and urine, respectively. Serum 3,5-T2 concentrations were neither associated with classical thyroid function parameters nor altered during experimental thyrotoxicosis. Strikingly, many metabolites related to coffee metabolism, including caffeine and paraxanthine, formed the clearest positively associated molecular signature. Importantly, these associations were replicated in the experimental human thyrotoxicosis model. Conclusion: The molecular fingerprint of 3,5-T2 demonstrates a clear and strong positive association of the serum levels of this TH metabolite with plasma levels of compounds indicating coffee consumption, therefore pointing to the liver as an organ, the metabolism of which is strongly affected by coffee. Furthermore, 3,5-T2 serum concentrations were found not to be directly TH dependent. Considering the beneficial health effects of 3,5-T2 administration observed in animal models and those of coffee consumption demonstrated in large epidemiological studies, one might speculate that coffee-stimulated hepatic 3,5-T2 production or accumulation represents an important molecular link in this connection.

Context: Thyroid hormone metabolites might affect the heart. The endogenous aminergic metabolite 3-iodothyronamine (T1am) reduces left ventricular ejection fraction (LVEF) in rodents., Objective: To investigate concentration of T1am and its association with LVEF and biomarkers of heart function in patients with chronic heart failure (CHF) without thyroid disease, including patients with cardiac cachexia (nonedematous weight loss >5% over 6 months)., Methods: Cross-sectional study. CHF was characterized by LVEF <45% and symptoms. Three groups were included (n = 19 in each group, matched on age, sex, and kidney function): patients with cachexia (CAC), patients without (non-CAC), and control (C) patients with prior myocardial infarction and LVEF >45%. T1am was measured by a monoclonal antibody-based chemiluminescence immunoassay. N-amino terminal pro-BNP (NT-proBNP) concentrations were also analyzed., Results: Mean (SD) LVEF: CAC, 32 ± 9%; non-CAC, 38 ± 8%; and C, 60 ± 8% (P < 0.0001). TSH, T4, and T3 levels did not differ between groups and did not correlate to T1am. Serum T1am (nmol/L) concentrations were higher in CHF: CAC (mean ± SD), 12.4 ± 6.6; non-CAC, 9.1 ± 5; and C, 7.3 ± 2.9. A negative association between T1am and LVEF was present after adjusting for sex, age, T3, and estimated glomerular filtration rate (P = 0.03). Further, serum T1am levels tended to be associated with NT-proBNP (P = 0.053)., Conclusion: Serum T1am levels were increased in patients with CHF and numerically highest (although nonsignificant) in patients with cardiac cachexia. Increasing T1am concentrations were independently associated with reduced LVEF, suggesting a direct effect on the human heart., (Copyright © 2019 Endocrine Society.)

Purpose: 3-iodothyronamine (3-T 1 AM), a decarboxylated and deiodinated thyroid hormone metabolite, leads at pharmacological doses to hypoinsulinemia, hyperglucagonemia and hyperglycemia in vivo. As the pancreatic Langerhans islets express thyroid hormone transmembrane transporters (THTT), we tested the hypothesis that not only plasma membrane-mediated 3-T 1 AM binding to and activation of G-protein coupled receptors, but also 3-T 1 AM metabolite(s) generated by 3-T 1 AM uptake and metabolism might decrease glucose-stimulated insulin secretion (GSIS)., Methods: Murine pancreatic β-cells MIN6 were characterized for gene expression of THTT, deiodinases and monoamine oxidases. 3-T 1 AM uptake and intracellular metabolism to the corresponding 3-iodothyroacetic acid were analysed by liquid-chromatography tandem mass spectrometry (LC-MS/MS) at different time points in cells as well as the conditioned medium. Mitochondrial activity, especially ATP-production, was monitored real-time after 3-T 1 AM application using Seahorse Bioanalyzer technique. Effect of 3-T 1 AM on GSIS into the culture medium was assayed by ELISA., Results: MIN6 cells express classical THTT, proposed to transport 3-T 1 AM, as well as 3-T 1 AM metabolizing enzymes comparable to murine primary pancreatic islets. 3-T 1 AM accumulates in MIN6 cells and is metabolized by intracellular MaoB to 3-iodothyroacetic, which in turn is rapidly exported. 3-T 1 AM decreases mitochondrial ATP-production concentration dependently. GSIS is diminished by 3-T 1 AM treatment. Using LC-MS/MS, no further 3-T 1 AM metabolites except 3-iodothyroacetic were detectable., Conclusions: This data provides a first link between cellular 3-T 1 AM uptake and regulation of mitochondrial energy metabolism in ß-cells, resulting in reduced insulin secretion. We conclude that MIN6 is an appropriate cell model to study 3-T 1 AM-dependent (intra-)cellular biochemical mechanisms affecting insulin production in vitro., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: 3-Iodothyronamine (3-T 1 AM) is an endogenous decarboxylated thyroid hormone (TH) metabolite. Pharmacological doses of 3-T 1 AM decrease heart rate, body temperature, and metabolic rate in rodents-effects that are contrary to classic TH excess. Furthermore, a single dose of 3-T 1 AM was shown to suppress the hypothalamic-pituitary-thyroid (HPT) axis in rats. It was hypothesized that 3-T 1 AM might play a role in the fine-tuning of TH action and might have a direct regulatory effect on the thyroid gland., Methods: This study tested whether repeated 3-T 1 AM treatment interfered with thyroid function and the HPT axis in mice. Therefore, male C57BL/6 mice were intraperitoneally injected with 5 mg/kg of 3-T 1 AM or vehicle daily for seven days. Additionally, the effects of 3-T 1 AM on the differentiated rat thyrocyte cell line PCCL3 were analyzed., Results: Repeated administration of 3-T 1 AM decreased thyroidal mRNA content of the sodium iodide symporter (Nis), thyroglobulin, and pendrin in mice. No interference with the HPT axis was observed, as determined by unaltered pituitary mRNA levels of triiodothyronine-responsive genes, including thyrotropin subunit β. Furthermore, 3-T 1 AM treatment did not change transcript levels of hepatic triiodothyronine-responsive genes, such as deiodinase 1. In line with this, serum TH concentrations were not changed after the treatment period of seven days. In concordance with the in vivo findings, 3-T 1 AM decreased the thyrotropin-dependent expression of Nis and functional iodide uptake in PCCL3 cells in vitro. Additionally, uptake and metabolism of 3-T 1 AM by PCCL3 cells was observed, as well as 3-T 1 AM-dependent changes in intracellular Ca 2+ concentration that might be involved in mediating the reported effects., Conclusions: In conclusion, 3-T 1 AM application decreased expression of selected TH synthesis genes by acting directly on the thyroid gland, and it might therefore affect TH synthesis without involvement of the HPT axis.

Background: Critical illness is hallmarked by low circulating thyroxine (T4) and triiodothyronine (T3) concentrations, in the presence of elevated reverse T3 (rT3) and low-normal thyrotropin (TSH), referred to as nonthyroidal illness (NTI). Thyroid hormone (TH) metabolism is substantially increased during NTI, in part explained by enhanced deiodinase 3 (D3) activity. T4- and T3-sulfate concentrations are elevated, due to suppressed D1 activity in the presence of unaltered sulfotransferase activity, and 3,3'-diiodothyronine (3,3'-T2) concentrations are normal. To elucidate further the driving forces behind increased TH metabolism during NTI, two other potential T4 metabolites-3,5-diiodothyronine (3,5-T2) and 3-iodothyronamine (3-T1AM)-were measured and related to their potential TH precursors., Methods: Morning blood samples were collected cross-sectionally from 83 critically ill patients on a University Hospital intensive care unit and from 38 demographically matched healthy volunteers. Serum TH and binding proteins were quantified with commercial assays, and 3,5-T2 and 3-T1AM with in-house developed immunoassays., Results: Critically ill patients revealed, besides the NTI, a median 44% lower serum 3-T1AM concentration (p < 0.0001) and a 30% higher serum 3,5-T2 concentration (p = 0.01) than healthy volunteers did. Non-survivors and patients diagnosed with sepsis upon admission to the intensive-care unit had significantly higher 3,5-T2 (p ≤ 0.01) but comparable 3-T1AM (p > 0.2) concentrations than other patients did. Multivariable linear regression analysis adjusted for potential precursors revealed that the reduced serum 3-T1AM was positively correlated with the low serum T3 (p < 0.001) but unrelated to serum T4 or rT3. The elevated 3,5-T2 concentration did not independently correlate with TH., Conclusions: Increased TH metabolism during NTI could not be explained by increased conversion to 3-T1AM, as circulating 3-T1AM was suppressed in proportion to the concomitantly low T3 concentrations. Increased conversion of T4 and/or T3 to 3,5-T2 could be possible, as serum 3,5-T2 concentrations were elevated. Whether 3-T1AM or 3,5-T2 plays a functional role during critical illness needs further investigation.

Objectives: Our aim was to investigate the association of thyroid function defined by serum concentrations of thyroid-stimulating hormone (TSH) with thoracic aortic wall thickness (AWT) as a marker of atherosclerotic processes., Methods: We pooled data of 2,679 individuals from two independent population-based surveys of the Study of Health in Pomerania. Aortic diameter and AWT measurements were performed on a 1.5-T MRI scanner at the concentration of the right pulmonary artery displaying the ascending and the descending aorta., Results: TSH, treated as continuous variable, was significantly associated with descending AWT (β = 0.11; 95 % confidence interval (CI) 0.02-0.21), while the association with ascending AWT was not statistically significant (β = 0.20; 95 % CI -0.01-0.21). High TSH (>3.29 mIU/L) was significantly associated with ascending (β = 0.12; 95 % CI 0.02-0.23) but not with descending AWT (β = 0.06; 95 % CI -0.04-0.16). There was no consistent association between TSH and aortic diameters., Conclusions: Our study demonstrated that AWT values increase with increasing serum TSH concentrations. Thus, a hypothyroid state may be indicative for aortic atherosclerosis. These results fit very well to the findings of previous studies pointing towards increased atherosclerotic risk in the hypothyroid state., Key Points: • Serum TSH concentrations are positively associated with aortic wall thickness. • Serum TSH concentrations are not associated with the aortic diameters. • Serum 3,5-diiodothyronine concentrations may be positively associated with aortic wall thickness.

The endogenous metabolite 3-iodothyronamine (3-T1AM) induces strong hypothermia and bradycardia at pharmacological doses. Although its biosynthesis from thyroid hormone precursors appears likely, the sequence and sites of reactions are still controversial: studies in T4-substituted thyroid cancer patients lacking functional thyroid tissue suggested extrathyroidal 3-T1AM production, whereas studies using labeled T4 in mice indicated intrathyroidal formation. However, because the patients received T4 orally, whereas the mice were injected ip, we hypothesized that 3-T1AM synthesis requires the intestinal passage of T4. Using the everted gut sac model in combination with mass spectrometry, we demonstrate 3-T1AM production from T4 in mouse intestine via several deiodination and decarboxylation steps. Gene expression analysis confirmed the expression of all 3 deiodinases as well as ornithine decarboxylase (ODC) in intestine. Subsequent experiments employing purified human ODC revealed that this enzyme can in fact mediate decarboxylation of 3,5-T2 and T4 to the respective thyronamines (TAMs), demonstrating that the intestine expresses the entire molecular machinery required for 3-T1AM biosynthesis. Interestingly, TAM production was strongly affected by the antithyroid treatment methimazole and perchlorate independently of thyroid status, limiting the validity of the respective mouse models in this context. Taken together, our data demonstrate intestinal 3-T1AM biosynthesis from T4 involving decarboxylation through ODC with subsequent deiodination, and explain the apparent discrepancy between 3-T1AM serum levels in patients substituted orally and mice injected ip with T4. Identifying ODC as the first enzyme capable of decarboxylating thyroid hormone, our findings open the path to further investigations of TAM metabolism on molecular and cellular levels.

Background: There is only limited data on the potential association between thyroid dysfunction and peripheral arterial disease (PAD)., Objective: The aim of our study was to investigate the potential association of thyroid function, as defined by serum concentrations of the clinically used primary thyroid function marker thyrotropin [i.e. thyroid-stimulating hormone (TSH)] and 3,5-diiodothyronine (3,5-T2), with the ankle-brachial index (ABI) as a marker of PAD., Methods: We used data from 5,818 individuals from three cross-sectional population-based studies conducted in Northeast (SHIP-2 and SHIP-TREND) and Central Germany (CARLA). Measurement of serum TSH concentrations was conducted in one central laboratory for all three studies. In a randomly selected subpopulation of 750 individuals of SHIP-TREND, serum 3,5-T2 concentrations were measured with a recently developed immunoassay. ABI was measured either by a hand-held Doppler ultrasound using the Huntleigh Dopplex D900 or palpatorily by the OMRON HEM-705CP device., Results: Serum TSH concentrations were not significantly associated with ABI values in any of the three studies. Likewise, groups of individuals with a TSH <0.3 mIU/l or with a TSH ≥3.0 mIU/l had no significantly different ABI values in comparison with individuals with a TSH in the reference range. Analyses regarding TSH within the reference range or serum 3,5-T2 concentrations did not reveal consistent significant associations with the ABI. No sex-specific associations were detected., Conclusions: The results of our study do not substantiate evidence for an association between thyroid function and PAD, but further studies are needed to investigate the associations of overt forms of thyroid dysfunction with PAD.

Context: 3,5-Diiodo-L-thyronine (3,5-T2) is a thyroid hormone metabolite which exhibited versatile effects in rodent models, including the prevention of insulin resistance or hepatic steatosis typically forced by a high-fat diet. With respect to euthyroid humans, we recently observed a putative link between serum 3,5-T2 and glucose but not lipid metabolism., Objective: The aim of the present study was to widely screen the urine metabolome for associations with serum 3,5-T2 concentrations in healthy individuals., Study Design and Methods: Urine metabolites of 715 euthyroid participants of the population-based Study of Health in Pomerania (SHIP-TREND) were analyzed by (1)H-NMR spectroscopy. Multinomial logistic and multivariate linear regression models were used to detect associations between urine metabolites and serum 3,5-T2 concentrations., Results: Serum 3,5-T2 concentrations were positively associated with urinary levels of trigonelline, pyroglutamate, acetone and hippurate. In detail, the odds for intermediate or suppressed serum 3,5-T2 concentrations doubled owing to a 1-standard deviation (SD) decrease in urine trigonelline levels, or increased by 29-50% in relation to a 1-SD decrease in urine pyroglutamate, acetone and hippurate levels., Conclusion: Our findings in humans confirmed the metabolic effects of circulating 3,5-T2 on glucose and lipid metabolism, oxidative stress and enhanced drug metabolism as postulated before based on interventional pharmacological studies in rodents. Of note, 3,5-T2 exhibited a unique urinary metabolic profile distinct from previously published results for the classical thyroid hormones.

Background: Although hyperthyroidism predisposes to atrial fibrillation, previous trials have suggested decreased triiodothyronine (T3) concentrations to be associated with postoperative atrial fibrillation (POAF). Therapy with thyroid hormones (TH), however, did not reduce the risk of POAF. This study reevaluates the relation between thyroid hormone status, atrial electromechanical function and POAF., Methods: Thirty-nine patients with sinus rhythm and no history of atrial fibrillation or thyroid disease undergoing cardiac surgery were prospectively enrolled. Serum concentrations of thyrotropin, free (F) and total (T) thyroxine (T4) and T3, reverse (r)T3, 3-iodothyronamine (3-T1AM) and 3,5-diiodothyronine (3,5-T2) were measured preoperatively, complemented by evaluation of echocardiographic and electrophysiological parameters of cardiac function. Holter-ECG and telemetry were used to screen for POAF for 10 days following cardiac surgery., Results: Seven of 17 patients who developed POAF demonstrated nonthyroidal illness syndrome (NTIS; defined as low T3 and/or low T4 syndrome), compared to 2 of 22 (p < 0.05) patients who maintained sinus rhythm. In patients with POAF, serum FT3 concentrations were significantly decreased, but still within their reference ranges. 3,5-T2 concentrations directly correlated with rT3 concentrations and inversely correlated with FT3 concentrations. Furthermore, 3,5-T2 concentrations were significantly elevated in patients with NTIS and in subjects who eventually developed POAF. In multivariable logistic regression FT3, 3,5-T2, total atrial conduction time, left atrial volume index and Fas ligand were independent predictors of POAF., Conclusion: This study confirms reduced FT3 concentrations in patients with POAF and is the first to report on elevated 3,5-T2 concentrations in cardiac NTIS. The pathogenesis of NTIS therefore seems to involve more differentiated allostatic mechanisms.

Background: During the last two decades, it has become obvious that 3,5-diiodothyronine (3,5-T2), a well-known endogenous metabolite of the thyroid hormones thyroxine (T4) or triiodothyronine (T3), not only represents a simple degradation intermediate of the former but also exhibits specific metabolic activities. Administration of 3,5-T2 to hypothyroid rodents rapidly stimulated their basal metabolic rate, prevented high-fat diet-induced obesity as well as steatosis, and increased oxidation of long-chain fatty acids., Objective: The aim of the present study was to analyze associations between circulating 3,5-T2 in human serum and different epidemiological parameters, including age, sex, or smoking, as well as measures of anthropometry, glucose, and lipid metabolism., Methods: 3,5-T2 concentrations were measured by a recently developed immunoassay in sera of 761 euthyroid participants of the population-based Study of Health in Pomerania. Subsequently, analysis of variance and multivariate linear regression analysis were performed., Results: Serum 3,5-T2 concentrations exhibited a right-skewed distribution, resulting in a median serum concentration of 0.24 nM (1st quartile: 0.20 nM; 3rd quartile: 0.37 nM). Significant associations between 3,5-T2 and serum fasting glucose, thyrotropin (TSH), as well as leptin concentrations were detected (p<0.05). Interestingly, the association to leptin concentrations seemed to be mediated by TSH. Age, sex, smoking, and blood lipid profile parameters did not show significant associations with circulating 3,5-T2., Conclusion: Our findings from a healthy euthyroid population may point toward a physiological link between circulating 3,5-T2 and glucose metabolism.

Background: Recent studies have shown associations of hypothyroidism with arterial blood pressure, atherosclerosis, and inflammation. Based on these pathways, there might also be an association between hypothyroidism and retinal arteriolar narrowing (RAN), a marker of microvascular damage from hypertension, atherosclerosis, and inflammation. Against this background, the aim of this study was to investigate the putative association between serum thyrotropin (TSH) levels and RAN defined by arterio-venous ratio (AVR) from static vessel analysis., Methods: We used data from 3189 individuals from the second population-based cohort of the Study of Health in Pomerania (SHIP-TREND-0). Thyroid function was defined according to serum TSH and serum diiodothyronine (3,5-T2) levels. Low and high serum TSH levels were defined by the cutoffs 0.3 mIU/L and 3.0 mIU/L. Fundus photography of the central retina was recorded with a nonmydriatic camera, and images were evaluated by one experienced reader. An AVR <0.8 was defined as decreased. Serum TSH levels, low and high TSH, and serum 3,5-T2 levels were associated with AVR by linear regression and with AVR <0.8 by Poisson regression, both adjusted for age, sex, cigarette smoking, alcohol consumption, and intake of beta-blockers., Results: Serum TSH levels were significantly associated with AVR (β=-0.028 [CI -0.049 to -0.007]; p=0.009) and with a decreased AVR <0.8 (relative risk=2.05 [CI 1.13-3.73]; p=0.019). Individuals with high TSH had a 1.43 higher risk for a decreased AVR ([CI 1.04-1.96]; p=0.027) than individuals with serum TSH levels within the reference range. Serum 3,5-T2 levels were also associated with a decreased AVR (relative risk for an increase of 1 nM=0.45 [CI 0.23-0.87]; p=0.017)., Conclusions: Our results substantiate evidence for an association between hypothyroidism and RAN. Potential mechanisms explaining this association are long-term hypertension, atherosclerotic processes, and inflammation.

Background: 3,5-Diiodo-L-thyronine (3,5-T2), a potential metabolite of 3,3',5-triiodothyronine (T3), exerts marked metabolic actions without the undesirable cardiac and central side effects of T3. So far the lack of reliable quantification methods for endogenous 3,5-T2 in human serum has limited further insight into its physiological and pathophysiological roles in endocrine homeostasis and disease status., Methods: Monoclonal anti-3,5-T2 antibodies (3,5-T2 mAbs) were produced in mice. We developed a competitive chemiluminescence immunoassay (CLIA) with one selected mAb and optimized it for high sensitivity, linearity, recovery, and low cross-reactivity to structurally related thyroid hormones (THs) and thyronamines. The CLIA was then used to investigate the origin and action of 3,5-T2 in humans under physiological and pathophysiological conditions in comparison with THs. Patient analysis included individuals with confirmed hypo- or hyperthyroidism and a separate population of thyroidectomized patients on L-thyroxine (T4) replacement therapy., Results: 3,5-T2 is stable in human serum after storage at 4°C or room temperature as well as several freeze-thaw cycles. The immunoassay did not show any significant cross-reactivity with naturally occurring TH metabolites in physiological and pathophysiological concentrations. The assay shows a lower detection limit of 0.2 nM 3,5-T2 and an upper detection limit of 10.0 nM. The newly established CLIA generates reliable results after spiking exogenous 3,5-T2 or by linear dilution of sera. Intra-assay variation is between 4.1% and 9.0%. Overall mean of variation between different assays is 5.6%-12.9%. 3,5-T2 serum concentrations do not differ in hyperthyroid (0.31 ± 0.02 nM, n=24) compared to hypothyroid (0.43 ± 0.04 nM, n=31) individuals. 3,5-T2 was detectable and elevated in serum from thyroidectomized and T4-substituted patients (0.48 ± 0.03 nM, n=100) in comparison to a sex- and age-matched control group (0.29 ± 0.01 nM, n=99)., Conclusion: The established CLIA is highly specific, sensitive, precise and accurate for 3,5-T2 detection in human serum. Because 3,5-T2 is not regulated in conditions of an altered thyroid state, it is most likely that serum 3,5-T2 concentrations are not directly dependent on feedback regulation via the hypothalamic-pituitary axis. In addition 3,5-T2 is present in thyroidectomized individuals on T4 substitution, and it is elevated after T4 substitution compared with healthy controls. We conclude that these data support extrathyroidal production of 3,5-T2 from T4.