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Cited by 23Thyroid Disorders and TreatmentsHormonal and reproductive studiesThyroid Cancer Diagnosis and Treatment

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

Open access
Köhrle, Josef, Lehmphul, Ina, Pietzner, Maik, Renko, Kostja, Rijntjes, Eddy, Richards, Keith, Anselmo, João, Danielsen, Mark +1 more·2020·Frontiers in Endocrinology
DOI 10.3389/fendo.2019.00787PMC PMC6960127
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Abstract

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.

AI snapshot

Written from the paper’s full text.

TL;DR

3,5-T2 is presented as a Janus-faced thyroid hormone metabolite with T3-mimetic endocrine actions and tissue-resident/intracrine hepatic effects, but its human serum concentrations are highly method-dependent and controversial. The review synthesizes clinical, animal, cell, and analytical studies showing no clear evidence that 3,5-T2 is formed directly from T4/T3 in humans, while elevated concentrations occur in several disease states and abuse is discouraged because of HPT-axis suppression and cardiac risks.

Key findings

  • Serum 3,5-T2 measurements diverge markedly by method: polyclonal RIA reports ranged from 4 to almost 200 pM/L, while a monoclonal antibody-based CLIA gave a reference-population concentration around 290 pM; LC-MS/MS found most human sera below 5 pM (lower limit of quantification), and another LC-MS study reported 78 ± 9 pM, attributed by the authors to internal-standard contamination.
  • In the population-based SHIP-Trend cohort of 761 euthyroid participants, CLIA-measured 3,5-T2 had a median of 0.24 nM with right-skewed distribution; one third were below the limit of quantification but above the limit of detection, and concentrations showed no clear association with serum T4/T3 or TSH but were positively associated with urinary coffee-consumption metabolites including trigonelline, pyroglutamate, and hippurate.
  • Four human paradigms administering T4, T3, or T3 sulfate found no significant changes in 3,5-T2 or 3-T1AM serum concentrations; individual 3,5-T2 concentrations were remarkably stable over hours to days, with pronounced inter-individual differences.
  • Elevated 3,5-T2 was reported in sepsis, ICU non-survivors, post-operative atrial fibrillation, impaired renal function/dialysis, and oral T4 supplementation; rodent and cell data support hepatic accumulation, antisteatotic effects, TR binding, HPT-axis suppression, and adverse cardiac effects, while a 4-week randomized trial of the 3,5-T2 mimetic TRC-150094 (50 mg daily) found no evidence for improved insulin sensitivity, plasma free fatty acids, or intrahepatic triglyceride content.

Methods

  • Design: narrative review/hypothesis synthesis combining clinical experimental paradigms, a population cohort, animal and cell-culture models, and analytical method comparisons rather than a single primary study.
  • Data/sample: human sera from euthyroid volunteers (n = 12 receiving a single 50 μg T3 dose), hypothyroid patients (n = 18 receiving 15–60 μg T3), thyroidectomized patients, dialysis patients, ICU patients, POAF patients, and 761 euthyroid SHIP-Trend participants; rodent and cell models; immune-extraction experiments with monoclonal antibodies.
  • Main analysis: RIA, monoclonal antibody-based CLIA, LC-MS/MS and LC-LIT-MS3, 1H-NMR urine metabolomics, receptor binding/competition data, gene-expression and mitochondrial functional readouts, and cross-study comparison of 3,5-T2 concentrations under physiological and pathophysiological conditions.

Limitations

  • Stated analytical limitations: immunoassay results vary with antibodies, tracer synthesis, and standard purity; 3,5-T2 may bind unknown serum distributor proteins; CLIA and LC-MS/MS results remain discrepant and unresolved; LC-MS quantification faces matrix, recovery, isomer separation, and internal-standard contamination problems.
  • Stated study limitations: SHIP-Trend used anonymized sera so health status could not be traced; no residual serum was available to repeat some studies with LC-LIT-MS3; the coffee-metabolite association lacks a tested causal hypothesis; many rodent studies used severely hypothyroid models, did not measure circulating or tissue 3,5-T2 concentrations, and did not systematically control for adverse effects; Table 4 receptor comparisons are not systematically comparable.
  • Evident limitations: the paper is a narrative/hypothesis synthesis with unpublished data and anecdotal self-administration, so many conclusions rely on cross-study comparisons and require prospective confirmation.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.3389/fendo.2019.00787

Cites

105 of 105 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

36 of 103 cited sources checked · 9 supported · 1 flagged · accuracy not yet measured

  • Findingcitation

    Reported serum concentrations for 3,3′-T2 covered a very narrow concentration range between 36 and 68 pM.

    “Reported serum concentrations covered a very narrow concentration range between 36 and 68 pM for 3,3′-T2 (see Table 1) (23–29, 32–34).”
    Quote in the paperNot a substantial claim2 of 10 sources checked
    • cites [23] Richards, 2017, Avoiding the pitfalls when quantifying thyroid hormones and their metabolites using mass spectrometric methods: the role of quality assurance, Mol Cell Endocrinol source
    • cites [24] Richards, 2017, A validated LC-MS/MS method for cellular thyroid hormone metabolism: uptake and turnover of mono-iodinated thyroid hormone metabolites by PCCL3 thyrocytes, PLoS ONE. source

      The Materials and methods section describes 3,3′-T2 standards used to spike cell lysates, and Results Table 2 reports detection limits in cell extracts, not serum concentrations. Searches for serum 3,3′-T2 and the 36–68 pM range found no passage in our copy reporting that range. Page numbers were not available. (LLM, conf=0.72)

      Experimental — accuracy not yet measured
    • cites [25] Richards, 2019, A combined LC-MS/MS and LC-MS3 multi-method for the quantification of iodothyronines in human blood serum, Anal Bioanal Chem. source
    • cites [26] Gu, 2010, Development and validation of an isotope dilution tandem mass spectrometry method for the simultaneous quantification of 3-iodothyronamine, thyroxine, triiodothyronine, and 3,3'-diiodo-l-thyronine in humans, Clin Chem.

      Not checked in the free run.

    • cites [27] Jonklaas, 2014, 3,3'-Diiodothyronine concentrations in hospitalized or thyroidectomized patients: results from a pilot study, Endocr Pract source

      The Abstract’s Results report observed 3,3′-T2 concentrations from 2.01 to 45.8 pg/mL across four quartiles—a more than 20-fold span, not the narrow 36–68 pM range claimed. The Methods’ assay methodology also gives a different, narrower reference interval of 6.7–23 pg/mL; it is not presented as the full range of reported concentrations. Page numbers are unavailable in our copy. (LLM, conf=0.86)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [28] Moreno, 2002, Are the effects of T3 on resting metabolic rate in euthyroid rats entirely caused by T3 itself?, Endocrinology source
    • cites [29] Pinna, 2002, Concentrations of seven iodothyronine metabolites in brain regions and the liver of the adult rat, Endocrinology source
    • cites [32] Jaedig, 1982, The effect of starvation and refeeding with oral versus intravenous glucose on serum 3,5-,3,3'-and 3'-5'-diiodothyronine and 3'-monoiodothyronine, Acta Endocrinol. source
    • cites [33] Kirkegaard, 1981, A radioimmunoassay of serum 3,5-diiodothyronine, Acta Endocrinol. source
    • cites [34] Nishikawa, 1981, Age-related changes of serum 3,3'-diiodothyronine, 3',5'-diiodothyronine, and 3,5-diiodothyronine concentrations in man, J Clin Endocrinol Metab source
  • Premisecitation

    Extrathyroidal formation of 3,5-T2 has been supported by studies using radioimmunoassays based on poly- or monoclonal antibodies.

    “Most of these studies were feasible after the development of specific radioimmunoassays, based on poly- or monoclonal antibodies in the Seventies of the last century, and supported extrathyroidal formation of 3,5-T2, which had been previously demonstrated using radioisotope-labeled TH in humans, experimental animal models, and in vitro systems [see recent reviews: (3, 9)].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [3] Köhrle, 2018, Thyroid hormones and derivatives: endogenous thyroid hormones and their targets source
    • cites [9] Köhrle, 2019, The colorful diversity of thyroid hormone metabolites, Eur Thyroid J source

      The sections on 3,5-T2 and its serum concentrations report radioimmunoassays and a later monoclonal-antibody immunoassay; the latter found 3,5-T2 in patients lacking functional thyroid tissue, consistent with extrathyroidal formation. But the paper describes that monoclonal assay as chemiluminescent, not a radioimmunoassay, and treats the proposed site of formation as an assumption rather than an established result of those assays. The supplied copy gives no page numbers for these passages. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
  • Findingcitation

    In a group of intensive care unit (ICU) patients, non-survivors had significantly higher 3,5-T2 concentrations than those who survived.

    “Of interest was the observation that, in a group of intensive care unit (ICU) patients, non-survivors had significantly higher 3,5-T2 concentrations than those who survived (42).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [42] Langouche, 2016, Circulating 3-T1AM and 3,5-T2 in critically ill patients: a cross-sectional observational study, Thyroid source

      The Results section (“Circulating thyroid hormones, metabolites and binding proteins”; page not given) reports higher serum 3,5-T2 in ICU non-survivors than survivors. It does not explicitly establish statistical significance for that comparison; its explicit significance statement concerns ICU patients versus healthy controls. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Findingcitation

    Patients with post-operative atrial fibrillation (POAF) had higher 3,5-T2 concentration than a similar group of cardiology patients without POAF.

    “Patients with post-operative atrial fibrillation (POAF) also had higher 3,5-T2 concentration than a similar group of cardiology patients without POAF (41).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [41] Dietrich, 2015, Nonthyroidal illness syndrome in cardiac illness involves elevated concentrations of 3,5-diiodothyronine and correlates with atrial remodeling, Eur Thyroid J. source
  • Premisecitation

    The weight loss reported in the self-administered pilot volunteer study could not be supported by a randomized, placebo-controlled, double-blind, 4-week trial on male cardio-metabolic patients receiving 50 mg daily TRC-150094.

    “This observation could not be supported by a randomized, placebo-controlled, double-blind, 4-week trial on male cardio-metabolic patients, who received a 50 mg daily dose of TRC-150094 (98).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [98] van der Valk, 2014, The effect of a diiodothyronine mimetic on insulin sensitivity in male cardiometabolic patients: a double-blind randomized controlled trial, PLoS ONE source
  • Classified as probably not a substantial claim. 19
  • May combine two claims. 21
  • The quote only partly states this claim. 1

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    T4 also binds to cell membrane-located ανβ3 integrin receptors which exert rapid signaling via various kinase and intracellular pathways, especially in tumor cells and stem cells.

    “Over the last years, some evidence has been presented that T4 also binds to cell membrane-located ανβ3 integrin receptors which exert rapid signaling via various kinase and intracellular pathways, especially in tumor cells and stem cells (7, 8).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [7] Davis, 2018, Bioactivity of thyroid hormone analogs at cancer cells, Front Endocrinol (Lausanne). source

      The Introduction identifies a cell-surface thyroid-hormone receptor on integrin αvβ3, and “Bioactivity of T4 at αvβ3” describes T4-mediated MAPK/ERK1/2 and PI3K signaling relevant to cancer cells. The cited passages discuss cancer cells and rapidly dividing endothelial cells, but do not establish the claim’s specific emphasis on stem cells; a search for “stem cells” found no supporting passage. No page numbers are available in our copy. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [8] Schmohl, 2019, Tetrac as an anti-angiogenic agent in cancer, Endocr Relat Cancer source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Tetrac, a deaminated side chain metabolite of T4, present in human serum at concentrations similar to those of T3, antagonizes T4 (and also T3) actions at the integrin receptor signaling.

    “The T4 metabolite Tetrac, a deaminated side chain metabolite, present in human serum at concentrations similar to those of T3 (3, 9) antagonizes such T4 (and also T3) actions at the integrin receptor signaling.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [3] Köhrle, 2018, Thyroid hormones and derivatives: endogenous thyroid hormones and their targets source
    • cites [9] Köhrle, 2019, The colorful diversity of thyroid hormone metabolites, Eur Thyroid J source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    T3 exerts the majority of known thyroid hormone effects at the tissue and cellular level, including hypothalamic and pituitary negative feedback regulation of the HPTP axis.

    “T3 exerts the majority of known thyroid hormone (TH) effects at the tissue and cellular level, including hypothalamic and pituitary negative feedback regulation of the hypothalamus-pituitary-thyroid-periphery (HPTP) axis (4–6).”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [4] Fonseca, 2013, Coordination of hypothalamic and pituitary T3 production regulates TSH expression, J Clin Invest source

      The Introduction (p. 1) calls T3 the biologically active thyroid hormone and says T3 negatively regulates TRH and TSHB, supporting the hypothalamic and pituitary feedback portion. It does not establish that T3 exerts the majority of known thyroid hormone effects across tissues and cells. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [5] Mendoza, 2017, New insights into thyroid hormone action, Pharmacol Ther source
    • cites [6] Louzada, 2018, Similarities and differences in the peripheral actions of thyroid hormones and their metabolites, Front Endocrinol source

      The section ‘Thyroid hormones, their metabolites and the hypothalamus-pituitary-thyroid axis’ (page not available) states that T3 reduces hypothalamic TRH and pituitary TSH secretion, supporting the negative-feedback claim. The ‘Genomic and non-genomic actions’ section describes T3’s cellular mechanism, but the copy does not establish that T3 exerts the majority of all known thyroid-hormone effects. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    L-T4 is solely produced by the thyroid gland and serves as a prohormone, while T3 is in part secreted by the thyroid gland and mainly generated in extra-thyroidal tissues by either type 1 or type 2 deiodinase (DIO1, DIO2).

    “A century of thyroxine research has led to the commonly held opinion in the thyroid hormone community that 3,3′,5,5′-tetraiodo-L-thyronine (L-Thyroxine, L-T4), which is solely produced by the thyroid gland, serves as a prohormone while T3 (3,3′5-triiodo-L-thyronine), in part secreted by the thyroid gland, is mainly generated in various extra-thyroidal tissues by either type 1 or type 2 deiodinase”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [1] Bianco, 2018, The deiodinase trio and thyroid hormone signaling, Methods Mol Biol source

      The Introduction says T4 is the main hormone produced by the thyroid gland, is minimally active, and is converted to T3 mostly outside the gland. The Fundamental Aspects section identifies D1 as a contributor to extrathyroidal T3 production, and the Abstract identifies D2 as converting T4 to T3. Our copy does not establish that the thyroid is the sole source of T4 or explicitly state that it secretes some T3. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [2] Köhrle, 2017, A century of thyroid hormone research - Vol. I: the expanded thyroid hormone network: novel metabolites and modes of action, Mol Cell Endocrinol source
    • cites [3] Köhrle, 2018, Thyroid hormones and derivatives: endogenous thyroid hormones and their targets source

  • May combine two claims.

    Premisecitation

    3,5-T2, in addition to its thyromimetic action at the classical T3 receptors at high concentrations, exerts rapid direct effects on mitochondria.

    “Various groups have demonstrated that 3,5-T2, in addition to its thyromimetic action at the classical T3 receptors at high concentrations, exerts rapid direct effects on mitochondria (6, 16–19)”
    Quote in the paperSubstantial claim1 of 5 sources checked
    • cites [6] Louzada, 2018, Similarities and differences in the peripheral actions of thyroid hormones and their metabolites, Front Endocrinol source

      In “Genomic and non-genomic actions of thyroid hormones” (page unavailable), the review says 3,5-T2 interacts weakly with classical thyroid hormone receptors compared with T3, consistent with action at higher concentrations. It also identifies mitochondria as a target, but says 3,5-T2 *may* stimulate respiration through direct mitochondrial action; the citing sentence presents that direct effect as established. The “Regulation of oxygen consumption” section (page unavailable) supports the rapid-effect component. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [16] Horst, 2005, Rapid stimulation of hepatic oxygen consumption by 3,5-di-iodo-L-thyronine, Biochem J source
    • cites [17] Goglia, 2005, Biological effects of 3,5- diiodothyronine (T2) source
    • cites [18] Lombardi, 2012, Responses of skeletal muscle lipid metabolism in rat gastrocnemius to hypothyroidism and iodothyronine administration: a putative role for FAT/CD36, Am J Physiol Endocrinol Metab source
    • cites [19] Moreno, 2017, Direct and rapid effects of 3,5-diiodo-L-thyronine (T2), Mol Cell Endocrinol source

  • May combine two claims.

    Premisecitation

    The clinical trial of TRC-150094 provided no evidence for increased insulin sensitivity, changes in plasma free fatty acids, or intra-hepatic triglyceride content.

    “Different from this promising animal experiment, the clinical trial provided no evidence for increased insulin sensitivity, changes in plasma free fatty acids, or intra-hepatic triglyceride content after administration of this agent (98).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [98] van der Valk, 2014, The effect of a diiodothyronine mimetic on insulin sensitivity in male cardiometabolic patients: a double-blind randomized controlled trial, PLoS ONE source

  • May combine two claims.

    Premisecitation

    Several laboratories have developed procedures to accurately determine total T4 and total T3 in human plasma or serum and largely agree on concentration ranges, but this consensus has not been achieved for most other TH metabolites.

    “While meanwhile several laboratories developed procedures to accurately determine total T4 and total T3 in human plasma or serum and largely agree on concentrations ranges (25, 67–73), this consensus has not been achieved yet for most of the other TH metabolites, which occur at much lower concentrations.”
    Quote in the paperSubstantial claim4 of 8 sources checked
    • cites [25] Richards, 2019, A combined LC-MS/MS and LC-MS3 multi-method for the quantification of iodothyronines in human blood serum, Anal Bioanal Chem. source
    • cites [67] Welsh, 2017, Assessment of thyroid function in intensive care unit patients by liquid chromatography tandem mass spectrometry methods, Clin Biochem source

      The Methods (Laboratory methods; page unavailable) describe a validated LC-MSMS procedure for measuring total T3 and T4 in human plasma and give reference intervals. The paper does not establish agreement on concentration ranges across laboratories or address whether a consensus exists for most other thyroid hormone metabolites. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [68] Welsh, 2016, DEC diagnosis of endocrine disease: how reliable are free thyroid and total T3 hormone assays?, Eur J Endocrinol source

      The Emerging role of LC-MSMS section describes sensitive, precise methods for measuring thyroid hormones, and the Clinical implications section says LC-MSMS measurement is available at most major reference laboratories. The paper does not establish that laboratories largely agree on total T4 and total T3 concentration ranges, or compare that agreement with results for most other thyroid-hormone metabolites. It instead reports discrepancies between commonly used immunoassays and LC-MSMS, particularly at low concentrations. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [69] Bowerbank, 2019, A direct comparison of liquid chromatography-mass spectrometry with clinical routine testing immunoassay methods for the detection and quantification of thyroid hormones in blood serum, Anal Bioanal Chem source

      The Abstract describes a procedure for measuring T3 and T4 in human serum, and Results and discussion reports strong agreement between that method and routine immunoassays. But the paper measures *free*, not total, hormones and does not establish that multiple laboratories agree on concentration ranges for total T3 and T4. The citation is attached to this first clause, not the subsequent statement about other metabolites. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [70] Sakai, 2015, Correlation between serum levels of 3,3',5'-triiodothyronine and thyroid hormones measured by liquid chromatography-tandem mass spectrometry and immunoassay, PLoS ONE source

      The Introduction describes several studies measuring serum T4 and T3 and says rT3 occurs at extremely low levels that make quantification difficult. The Discussion reports strong T4 and T3 correlations for this study and another, but also notes measurement discrepancies. Neither section establishes agreement among laboratories on concentration ranges or a lack of consensus for most other thyroid-hormone metabolites beyond rT3. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [71] De Brabandere, 1998, Isotope dilution-liquid chromatography/electrospray ionization-tandem mass spectrometry for the determination of serum thyroxine as a potential reference method, Rapid Commun Mass Spectrom. source
    • cites [72] Tai, 2002, Candidate reference method for total thyroxine in human serum: use of isotope-dilution liquid chromatography-mass spectrometry with electrospray ionization, Clin Chem source
    • cites [73] Tai, 2004, Development and evaluation of a reference measurement procedure for the determination of total 3,3',5-triiodothyronine in human serum using isotope-dilution liquid chromatography-tandem mass spectrometry, Anal Chem source

  • May combine two claims.

    Premisecitation

    Moreno et al. observed that 3,5-T2 (25 μg/100 g body weight) exerted rapid effects in acutely treated hypothyroid rats while T3 reacted more slowly but with longer lasting effects.

    “They observed that 3,5-T2 (25 μg/100 g body weight) exerted rapid effects in acutely treated hypothyroid rats while T3 reacted more slowly but with longer lasting effects compared to 3,5-T2.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [90] Moreno, 1997, How the thyroid controls metabolism in the rat: different roles for triiodothyronine and diiodothyronines, J Physiol source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    No clear demonstration of rT3 function has been observed apart from its role during glial cell-mediated neuronal guidance in mammalian brain development.

    “Up to now, no clear demonstration of rT3 function has been observed apart from its role during glial cell-mediated neuronal guidance in mammalian brain development (11).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [11] Farwell, 2005, Regulation of cerebellar neuronal migration and neurite outgrowth by thyroxine and 3,3′,5′-triiodothyronine, Brain Res Dev Brain Res source

  • May combine two claims.

    Findingcitation

    Tissue levels of 3,5-T2, for example in the rat brain, were much higher than expected from serum concentrations and reached values in the range of those of T4 and T3.

    “Of interest was the observation that tissue levels of 3,5-T2, for example in the rat brain, were much higher than expected from serum concentrations and reached values in the range of those of T4 and T3, as clearly shown by Pinna et al. (29)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [29] Pinna, 2002, Concentrations of seven iodothyronine metabolites in brain regions and the liver of the adult rat, Endocrinology source

  • May combine two claims.

    Findingown data

    3,5-T2 concentrations in hypothyroid or hyperthyroid patients were not different from those of the reference population, while total T4 and T3 showed expected changes.

    “First of all, 3,5-T2 concentrations in hypothyroid or hyperthyroid patients were not different from those of the reference population, while total T4 and T3 showed expected changes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    We did not observe gender- or age-dependent variations in 3,5-T2 concentrations, nor were concentrations correlated with BMI.

    “We did not observe gender- or age-dependent variations, nor were concentrations correlated with BMI.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    One third of the SHIP-Trend study population had 3,5-T2 serum concentrations determined by CLIA below the limit of quantification but above the limit of detection, yielding a median serum concentration of 0.24 nM with right-skewed distribution.

    “Surprisingly, one third of the study population had 3,5-T2 serum concentrations determined by CLIA below the limit of quantification but above the limit of detection, yielding a median serum concentration of 0.24 nM with right-skewed distribution.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    High serum 3,5-T2 is associated with elevated urinary concentrations of metabolites related to coffee consumption.

    “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.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    The existence of 3,5-T2 in thyroglobulin has not been clearly demonstrated and appears biochemically improbable.

    “The existence of 3,5-T2 in thyroglobulin has not been clearly demonstrated (49, 50) and appears biochemically improbable considering that TPO catalyzes coupling of mono- and/or di-iodinated tyrosine residues to yield either T4 or T3, while no such coupling was observed between diiodotyrosine and iodine-free tyrosine in reports available.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [49] Alexander, 1979, Rapid analysis for iodotyrosines and iodothyronines in thyroglobulin by reversed-phase liquid chromatography, Clin Chem source
    • cites [50] Laurberg, 1985, Multisite inhibition by ipodate of iodothyronine secretion from perfused dog thyroid lobes, Endocrinology source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    One individual in that drug study was exceptional in exhibiting pronounced variations of 3,5-T2 for unknown reasons.

    “Only one individual was exceptional in exhibiting rather pronounced variations of 3,5-T2 for unknown reasons (unpublished data, van Vliet and Köhrle).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    Among these urinary metabolites are trigonelline, pyroglutamate, and hippurate.

    “Among these urinary metabolites are trigonelline, pyroglutamate, and hippurate.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The mAb against 3-T1AM extracted spiked 3-T1AM from serum, but no spiked 3,5-T2, no other THM, and not any endogenous 3-T1AM was found in the extracted eluates.

    “In contrast, the mAb against 3-T1AM, also coupled to beads, extracted spiked 3-T1AM from serum, but no spiked 3,5-T2, no other THM and surprisingly not any “endogenous” 3-T1AM was found in the extracted eluates (Table 3) (J.K. et al., unpublished data).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    The 3,5-T2 antibody efficiently extracted 3,5-T2 from serum but did not bind and extract spiked 3-T1AM or other endogenous THM.

    “Evaluation of extracts from mAb beads by LC-MS analysis (25) revealed that the 3,5-T2 antibody efficiently extracted 3,5-T2 from serum as expected but did not bind and extract spiked 3-T1AM or other endogenous THM.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [25] Richards, 2019, A combined LC-MS/MS and LC-MS3 multi-method for the quantification of iodothyronines in human blood serum, Anal Bioanal Chem. source

  • The quote only partly states this claim.

    Findingown data

    In healthy individuals and hypothyroid patients, 3,5-T2 serum concentrations differ markedly between individuals.

    “Most individuals exhibit very narrow concentration ranges with small variations over sampling time while individuals differ among each other in their 3,5-T2 concentration in serum.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    These urine metabolites represent a 'signature of coffee consumers.'

    “More detailed metabolome analyses revealed that these metabolites represent a “signature of coffee consumers.””
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Study design and conductnone given

    The CLIA was validated according to state-of-the-art technology with respect to assay stability, specificity, and reproducibility.

    “Using this assay, validated according to state-of-the-art technology with respect to assay stability, specificity, and reproducibility, we detected serum 3,5-T2 concentration in reference populations around 290 pM.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Thyroid hormone metabolites (THMs) are bioactive compounds.

    “Over the last decades, thyroid hormone metabolites (THMs) received marked attention as it has been demonstrated that they are bioactive compounds.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Premisenone given

    3,5-T2 has been considered the main biological active metabolite of T3, formed via further phenolic ring deiodination from T3.

    “3,5-T2 has been considered the main biological active metabolite of T3, formed via further phenolic ring deiodination from T3 (Figure 1).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    3,5-T2 turned out to be an ambiguous, Janus-type endogenous thyroid hormone metabolite with beneficial and adverse biological effects.

    “3,5-T2 turned out to be an ambiguous, Janus-type endogenous thyroid hormone metabolite (THM) with beneficial and adverse biological effects.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing). May combine two claims.

    Interpretationreasoning

    These mitochondrial effects might be beneficial in terms of stimulation of oxygen consumption, increased hepatic, and muscular lipid metabolism-all of these effects appear as potentially favorable in global attempts to combat steatosis in liver and other tissues.

    “which might be beneficial in terms of stimulation of oxygen consumption, increased hepatic, and muscular lipid metabolism—all of these effects appear as potentially favorable in global attempts to combat steatosis in liver and other tissues.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Horst et al. observed rapid stimulation of oxygen consumption in isolated perfused livers from hypothyroid rats by low picomolar 3,5-T2 within 90 min, while T4 and T3 reacted more slowly and their effect could be blocked by PTU.

    “The first evidence for rapid T3-independent effects of 3,5-T2 on mitochondrial activity and oxygen consumption was provided by Horst et al. (16), who observed rapid stimulation of oxygen consumption in isolated perfused livers, removed from hypothyroid rats.”
    • Also stated in: § 3,5-T2 Action on Liver Is Accompanied by Potentially Adverse Effects on HPT-Axis and Heart · plain
      “In their model, already low picomolar 3,5-T2 concentrations stimulated O2 consumption within 90 min, while T4 and T3 reacted more slowly and their effect could be blocked by adding 1 μM PTU to the perfusion medium”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [16] Horst, 2005, Rapid stimulation of hepatic oxygen consumption by 3,5-di-iodo-L-thyronine, Biochem J source
    • The authors state this as an assumption: DIO1 is the enzyme responsible for 3,5-T2 production.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationreasoning

    The effect of 3,5-T2 on hepatic metabolism, especially lipid metabolism, might be due to a remarkable accumulation of 3,5-T2 in the liver.

    “That effect of 3,5-T2 on hepatic metabolism, especially lipid metabolism, might be due to a remarkable accumulation of 3,5-T2 in the liver”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Pinna et al. determined 3,5-T2 concentrations in rat brains based on immunoassay under careful methodological precautions.

    “Pinna et al. (29) determined 3,5-T2 concentrations based on immunoassay under careful methodological precautions in rat brains”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [29] Pinna, 2002, Concentrations of seven iodothyronine metabolites in brain regions and the liver of the adult rat, Endocrinology source

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    It remains controversial whether 3,5-T2 would be an important lead compound in the development of antisteatotic drugs lacking unwanted adverse side effects at other TH responsive target tissues and reactions.

    “Thus, it remains quite controversial whether 3,5-T2 would be an important lead compound in the development of antisteatotic drugs lacking unwanted adverse side effects at other TH responsive target tissues and reactions.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The human studies indicate that 3,5-T2 is not directly generated from serum T3 and its serum profile does not mimic the transient increases observed for T3 after exogenous administration in humans.

    “Together, these studies might indicate that 3,5-T2 is not directly generated from serum T3 and its serum profile does not mimic the transient increases observed for T3 after its exogenous administration in humans.”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: T3 is a precursor of 3,5-T2.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premiseown data

    Assay accuracies, precisions, and process efficiencies with quality assessment parameters were obtained which are requested by authorities for analytical procedures utilized in clinical diagnostics.

    “Assay accuracies, precisions, and process efficiencies with quality assessment parameters were obtained which are requested by authorities for analytical procedures utilized in clinical diagnostics.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The method used by Richards et al. allowed clear separation of isobaric tri- and di-iodinated iodothyronines and resulted in acceptable signal-noise ratios for LIT-MS3 scans leading to limits of quantification in the low picomolar range.

    “This sample work-up was then combined with isotope dilution, LC-positive ion electron spray multiple reaction monitoring (MRM) linear ion trap LIT-MS3, which allowed clear separation of isobaric tri- and di-iodinated iodothyronines and resulted in acceptable signal-noise ratios for the LIT-MS3 scans leading to limits of quantification in the low picomolar range.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    3,5-T2, unlike its structural isomers 3,3′-T2 and 3′,5′-T2, is an active ligand for classical T3 receptors, binds with relatively high affinity, and modulates transcriptional activity of TR.

    “3,5-T2, in contrast to its structural isomers 3,3′-T2 and 3′,5′-T2, is an active ligand for the classical T3 receptors (TR), where it binds with relative high affinity (Table 4) and modulates transcriptional activity of TR”
    Quote in the paperSubstantial claim0 of 5 sources checked
    • cites [39] Jonas, 2015, 3,5-Diiodo-L-thyronine (3,5-T2) exerts thyromimetic effects on hypothalamus-pituitary-thyroid axis, body composition, and energy metabolism in male diet-induced obese mice, Endocrinology source
    • cites [81] Ball, 1997, 3,5-Diiodo-L-thyronine (T2) has selective thyromimetic effects in vivo and in vitro, J Mol Endocrinol source
    • cites [85] Horst, 1995, 3,5-Di-iodo-L-thyronine suppresses TSH in rats in vivo and in rat pituitary fragments in vitro, J Endocrinol. source
    • cites [86] Baur, 1997, 3,5-diiodo-L-thyronine stimulates type 1 5'deiodinase activity in rat anterior pituitaries in vivo and in reaggregate cultures and GH3 cells in vitro, Endocrinology source
    • cites [87] Lietzow, 2016, 3,5-T2 alters expression of murine genes relevant for xenobiotic, steroid, and thyroid hormone metabolism, J Mol Endocrinol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A group working with Goglia et al. presented evidence that 3,5-T2 might exert direct effects on mitochondria via interaction with electron transport chain proteins and F0-ATPase.

    “a group of researchers working with Goglia et al. over the last three decades presented evidence that 3,5-T2 might exert direct effects on mitochondria via interaction with proteins of the electron transport chain (e.g., cytochrome C oxidase) and with F0-ATPase [for review see (17)].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [17] Goglia, 2005, Biological effects of 3,5- diiodothyronine (T2) source
35 of 35

  • The paper relies on this without support.

    3,5-T2 occurs at low nanomolar concentrations in human serum.

    “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.”

  • The paper relies on this without support.

    3,5-T2 might reach tissue concentrations similar to those of T4 and T3, at least based on data from rodent models.

    “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.”

  • The paper relies on this without support.

    Commercial preparations of 3,5-T2 standards available now and in the past frequently were significantly contaminated with T3 in concentrations high enough to provide interference by T3 serum concentrations.

    “A similar problem exists also with respect to the purity of 3,5-T2 standards used in these immunoassays, as commercial preparations available now and in the past frequently were significantly contaminated with T3 in concentrations high enough to provide interference by T3 serum concentrations, which are typically an order of magnitude higher than those of 3,5-T2 in the 3 nM concentration range.”

  • The paper relies on this without support.

    3,5-T2 is found in blood and at even higher concentrations in several tissues.

    “The TH metabolite 3,5-T2 is found in blood and at even higher concentrations in several tissues.”

  • The paper relies on this without support.

    3,5-T2 has potency in suppressing the HPT axis and causing adverse cardiac side effects.

    “considering its potency in suppressing the HPT axis and causing adverse cardiac side effects.”

  • The authors state this as an assumption.

    T3 is a precursor of 3,5-T2.

    “Assuming T3 as precursor of 3,5-T2, one would expect an increase of 3,5-T2 concentration after T3 administration.”

  • The authors state this as an assumption.

    DIO1 is the enzyme responsible for 3,5-T2 production.

    “which acts as an inhibitor of DIO1, assumed to be the enzyme responsible for 3,5-T2 production.”

  • The paper relies on this without support.

    Adverse effects of 3,5-T2 observed in rodent hearts have not been studied in more detail.

    “The adverse effects observed in rodent hearts have not been studied in more detail”

  • The paper relies on this without support.

    3,5-T2 is highly popular in the body-building and wellness scene and freely available via the internet.

    “3,5-T2, highly popular in the body-building and wellness scene and freely available via the internet”
9 of 9

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Stated 3,3′-T2 concentration range excludes a value in Table 1

    Minor

    The text gives a range of 36-68 pM for reported 3,3′-T2 serum concentrations, but Table 1 includes a human-serum range of 12.2-28.2 pM.

  • Conflicting accounts of where most T3 is produced

    Minor

    The introduction says T3 is mainly generated in extra-thyroidal tissues, whereas the Figure 1 caption says approximately 80% is secreted by the thyroid gland.

  • 3,5-T2 is described both as unable and able to bind classical T3 receptors

    Minor

    The graphical abstract says the iodine substitution that 3,5-T2 lacks is essential for binding classical T3 receptors. The mechanism section instead states that 3,5-T2 binds those receptors with relatively high affinity.

  • Conflicting deiodination positions for T3-to-3,5-T2 formation

    Minor

    The Figure 1 caption identifies the proposed conversion of T3 to 3,5-T2 as 3′-deiodination, while the biosynthesis section calls it 5′-deiodination.

4 of 4

Cites

Flagged first, then by citations

1 of the 106 references couldn’t be matched to a known paper.

Research works printed but not linked: 4

  1. Gu, 2010, Development and validation of an isotope dilution tandem mass spectrometry method for the simultaneous quantification of 3-iodothyronamine, thyroxine, triiodothyronine, and 3,3'-diiodo-l-thyronine in humans, Clin Chem., 56, A60
  2. Rokos, 1981, Synthesis and investigation of substituted iodothyronines with the aim of obtaining radiochemically stable 125I-tracers of high specific activity (author's transl), J Clin Chem Clin Biochem., 19, 191
  3. LehmphulI 10.18452/17434Zelluläre Wirkung, Wirkmechanismen und Nachweisverfahren von Schilddrüsenhormonen und ihren Metaboliten2015 · DOI
  4. Oppenheimer, 1996, The molecular basis of thyroid hormone actions, 162

Cited by

Sorted by citations

Related

Authors
Köhrle, J., Lehmphul, I., Pietzner, M., Renko, K., Rijntjes, E., Richards, K., Anselmo, J., Danielsen, M., & Jonklaas, J.
Affiliations
  • Humboldt-Universität zu Berlin (DE)
  • University of Cambridge (GB)
  • Hospital do Divino Espírito Santo (PT)
  • Georgetown University (US)
Corresponding author
Köhrle, Josef
ORCID
Venue
Frontiers in Endocrinology
Publisher
Frontiers Media SA
Published
2020-01-08
Volume
10
Pages
787-787
ISSN
1664-2392 (ISSN-L)
DOI
10.3389/fendo.2019.00787
PMC
PMC6960127
OpenAlex
W2998793948
Semantic Scholar
e653189f8a0a0eb29fdfff3210ffecc4b478effa
Cited by
23
References
106
Citations by year
2025: 1 · 2024: 3 · 2023: 3 · 2022: 6 · 2021: 4 · 2020: 4 · 2018: 1 · 2017: 1
FWCI
3.19
Type
journal article
Language
en
Open access
Licence
Locations
  • Frontiers in Endocrinology (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Apollo (University of Cambridge) (submittedVersion, cc-by)
  • Apollo (cc-by)
Funders
Deutsche Forschungsgemeinschaft
Topics
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Thyroid Disorders and Treatments
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Hormonal and reproductive studies
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Thyroid Cancer Diagnosis and Treatment
Keywords
3,5-diiodothyronine, thyroid hormone metabolites, hepatic metabolism, LC-MS/MS quantification, autoimmune thyroid disease, postoperative atrial fibrillation, coffee metabolites

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-11-03.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-03.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

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