Recovery of 3-iodothyronamine and derivatives in biological matrices: problems and pitfalls.

Related Articles

Recovery of 3-iodothyronamine and derivatives in biological matrices: problems and pitfalls.

Thyroid. 2017 Aug 31;:

Authors: Lorenzini L, Ghelardoni S, Saba A, Sacripanti G, Chiellini G, Zucchi R

Abstract
BACKGROUND: Difficulties have been reported in quantitating 3-iodothyronamine (T1AM) in blood or serum, and tentatively attributed to problems in extraction or other pre-analytical steps. For this reason even cell culture experiments have often be performed with unphysiological protein-free media. The aim of this study was to evaluate the recovery of exogenous T1AM added to a standard cell culture medium, namely DMEM supplemented with fetal bovine serum (FBS), and to other biological matrices.
METHODS: Cell culture media (Krebs buffer, DMEM, FBS, DMEM+FBS, used either in the absence or in the presence of NG108-15 cells) and other biological matrices (rat brain and liver homogenates, human plasma and blood) were spiked with T1AM and/or deuterated T1AM (d4-T1AM) and incubated for times ranging from 0 to 240 min. Samples were then extracted using a liquid/liquid method and analyzed using liquid chromatography coupled to mass spectrometry, to assay T1AM and its metabolites, namely 3-iodothyroacetic acid (TA1), thyronamine, thyroacetic acid, N-acetyl-T1AM and T1AM esters.
RESULTS: In FBS-containing buffers, T1AM decreased exponentially over time, with half life of 6-17 min, depending on FBS content, and after 60 min it averaged 0-10% of the baseline. T1AM metabolites were not detected, except for minimum amounts of TA1. Notably, d4-T1AM decreased over time at a much lower rate, reaching 50-70% of the baseline at 60 min. These effects were completely abolished by protein denaturation and partly reduced by semicarbazide. In the presence of cells, T1AM concentration decreased virtually to zero within 60 min, but TA1 accumulated in the incubation medium, with quantitative recovery. Spontaneous decrease in T1AM concentration with isotopic difference was confirmed in rat organ homogenates and human blood.
CONCLUSIONS: Our results suggest binding and sequestration of T1AM and/or its aldehyde derivative by blood and tissue proteins, with significant isotope effects. These issues might account for the technical problems complicating the analytical assays of endogenous T1AM.

PMID: 28859548 [PubMed - as supplied by publisher]

from #ENT-PubMed via ola Kala on Inoreader http://ift.tt/2iPPA8M