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
