Detecting 3-iodothyronamine in the presence of fetal bovine serum: isotope kinetic effect and other pitfalls

Background: Difficulties have been reported in quantitating 3-iodothyronamine (T1AM) in blood or serum, and most in vitro studies have been performed in the absence of serum proteins. The aim of this study was to develop a method to measure T1AM in a standard cell culture medium, namely DMEM supplemented with fetal bovine serum (FBS), and to investigate potential complications caused by serum components. Methods: FBS and DMEM+10-50% FBS samples were spiked with T1AM and/or deuterated T1AM (T1AM-d4) at the concentration of 10 ng/ml and incubated between 0 and 2 hours. Samples were then extracted using a liquid/liquid method and analyzed using liquid chromatography coupled to mass spectrometry (LC-MS/MS). The catabolites thyronamine, 3-iodothyroacetic acid (TA1) and thyroacetic acid were also measured. Results: Within 1 hour incubation, T1AM signal decreased to 0.1-14% (depending on FBS content), with an half-life of 5-18 min, while T1AM-d4 signal decreased to 41-76% of the initial peak area. TA1 was detected and accounted about 10% of the missing T1AM, while other catabolites were not present. T1AM decrease was prevented by preincubating samples with 8 M urea or 0.3 mg/ml proteinase K, and reduced over 50% by 0.1 mM semicarbazide, while monoamine oxidase inhibitors were ineffective. Moreover, significant TA1 production was observed by providing NG108-15 cells with DMEM+FBS that had been preincubated for 2 hours, in which T1AM was no longer detectable. Conclusions: T1AM signal decreases exponentially in FBS- supplemented media, and the decay is much slower with T1AM-d4, suggesting a remarkable isotope kinetic effect. This phenomenon depends on serum proteins, and semicarbazide-sensitive proteins, possibly semicarbazide-sensitive amine oxidases, appear to play the major role. These issues should be taken in account to develop an effective method to assay T1AM in blood and in the usual cell culture media.