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. 2017 Aug 24;12(8):e0183482. doi: 10.1371/journal.pone.0183482. eCollection 2017.

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.

Publication types

  • Validation Study

MeSH terms

  • Cell Line
  • Chromatography, High Pressure Liquid / methods*
  • Hep G2 Cells
  • Humans
  • Iodine / metabolism*
  • Limit of Detection
  • Mass Spectrometry / methods*
  • Quality Control
  • Reproducibility of Results
  • Thyroid Gland / cytology
  • Thyroid Gland / metabolism*
  • Thyroid Hormones / metabolism*

Substances

  • Thyroid Hormones
  • Iodine

Grant support

This work was supported by the Deutsche Forschungsgemeinschaft DFG (KO922-16/1-2 to J.K. and KO922-17/1-2 to J.K) in the framework of the DFG funded Priority Programme SPP1629 THYROIDTRANSACT and a Charité thesis scholarship (to N.S.). The company Chromicent GmbH played no part in any capacity in the project funding, intellectual input or experimentation, nor in the decision to publish/preparation of the manuscript. Daniel Rathmann’s role (who now works for Chromicent GmbH) was carried out whilst still an employee at Charite Universitätsmedizin, Berlin. The DFG solely provided financial support in the form of authors' salaries and/or research materials, they had no other involvement.