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Cited by 15Thyroid Disorders and TreatmentsAnalytical Methods in PharmaceuticalsAnalytical chemistry methods development

A validated LC-MS/MS method for cellular thyroid hormone metabolism: uptake and turnover of mono-iodinated thyroid hormone metabolites by PCCL3 thyrocytes

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Richards, Keith H., Schanze, Nancy, Monk, Ray, Rijntjes, Eddy, Rathmann, Daniel, Köhrle, Josef·2017·PLOS ONE
DOI 10.1371/journal.pone.0183482PMC PMC5570372
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Abstract

Tyrosine and phenolic ring de-iodination of thyroid hormones (TH) is crucial for regulating their physiological activity. Furthermore, reactions such as de-carboxylation to thyronamines (TAM) and de-amination to thyroacetic acids (TAc) produce TH metabolites (THM) with distinct biological properties. This needs to be considered when studying effects of TH and THM. The accurate and precise quantitative analysis of TH and THM in cell culture supernatants and cell lysates are key procedures required for studying the in vitro metabolism of TH. We report here the development of a liquid-liquid extraction/isotope dilution-liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the quantification of 9 thyronines (TN) and 6 TAM in human hepatocellular carcinoma Hep G2 cell lysate extracts. In addition, we adapted the method to quantify TH, TAM and TAc, in cell lysates of FBS-depleted rat thyroid epithelium PCCL3 cells. The methods for both cell lines were validated by rigorous assessment of linearity, limits of quantification and detection (LLOQ and LLOD respectively), intra- and inter-day accuracy, precision, process efficiency (PE), matrix effect (ME) and relative recovery (RE). Calibration curves covering 11 concentrations (based on 400 μl of lysate) were linear in the range 0.016-50 nM and 0.010-50 nM for Hep G2 and PCCL3 cells respectively. The lower limits of quantification were in the range 0.031 to 1 nM. We applied the PCCL3 version of the LC-MS/MS method to the analysis of lysed cell extracts from PCCL3 cells that had been incubated with 3-iodo-L-thyronine (T1), 3-iodothyronamine (3-T1AM) and 3-iodothyroacetic acid (3-T1Ac). Over the course of 30 minutes incubation 3-T1AM was de-iodinated to 4-[4-(2-aminoethylphenoxy)]phenol (thyronamine, T0AM) and de-aminated to 3-T1Ac respectively, whilst T1 underwent de-iodination to T0. This data indicates avid metabolism of these mono-iodinated compounds and the utility of LC-MS/MS to quantify such cellular metabolism.

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  • 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).”

    From 3,5-T2—A Janus-Faced Thyroid Hormone Metabolite Exerts Both Canonical T3-Mimetic Endocrine and Intracrine Hepatic Action (2020) · cites this paper as [24]

    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

  • Reported concentrations of most other TH metabolites in human or experimental animal serum, plasma, and tissue show wide variations and marked insufficiencies in method validation, standardization, and quality assessment.

    “while reported concentrations in human or experimental animal serum, plasma, and tissue show wide variations and marked insufficiencies in terms of method validation, standardization, and quality assessment parameters [see (23, 24, 74)].”

    From 3,5-T2—A Janus-Faced Thyroid Hormone Metabolite Exerts Both Canonical T3-Mimetic Endocrine and Intracrine Hepatic Action (2020) · cites this paper as [24]

    The Abstract and Results describe validation and quality-control measurements for LC-MS/MS analysis of cell-culture extracts, not a comparison of reported metabolite concentrations in human or animal serum, plasma, and tissue. Searches for concentration variation and shortcomings in validation, standardization, and quality assessment found no passage in our copy making that broader claim. (LLM, conf=0.72)

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Cites

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Cited by

Sorted by citations

Authors
Richards, K. H., Schanze, N., Monk, R., Rijntjes, E., Rathmann, D., & Köhrle, J.
Affiliations
Charité – Universitätsmedizin Berlin (DE)
Corresponding author
Köhrle, Josef
ORCID
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2017-08-24
Volume
12
Issue
8
Pages
e0183482
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0183482
PMC
PMC5570372
OpenAlex
W2748125750
Cited by
15
References
2
Citations by year
2026: 2 · 2022: 3 · 2020: 4 · 2019: 3 · 2018: 2 · 2017: 1
FWCI
2.16
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Refubium (Universitätsbibliothek der Freien Universität Berlin) (submittedVersion, cc-by)
Funders
Deutsche Forschungsgemeinschaft (KO922/17-1/2, SPP1629)
Topics
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Thyroid Disorders and Treatments
  • Physical Sciences › Chemistry › Analytical Chemistry › Analytical Methods in Pharmaceuticals
  • Physical Sciences › Chemistry › Analytical Chemistry › Analytical chemistry methods development
Keywords
thyronamines, deamination, HepG2 cells, LC-MS/MS method validation, 3-iodothyronamine
MeSH terms
Cell Line, Chromatography, High Pressure Liquid, Humans, Iodine, Quality Control, Mass Spectrometry, Thyroid Gland, Thyroid Hormones, Reproducibility of Results, Hep G2 Cells, Limit of Detection

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