Endocrine Abstracts

3-iodothyronamine (T₁AM) is rapidly catabolized to iodothyroacetic acid after few hours by serum proteins. To avoid a rapid metabolism during any treatment, DMEM devoid of FBS has been used, inducing changes in the cell culture environmental. We evaluated the uptake and the metabolism of different concentrations of T1AM, in presence of supplemented medium, and the presence of 3-iodothyroacetic aldehyde (Ald), a putative intermediate of T1AM catabolism to TA1.

DMEM supplemented with 10% FBS was spiked with T1AM at concentration 0.1–10 µM and provided to NG108–15 cells or U-87 MG (24-well plates). The medium was removed at specific time points (1,2,3,4, 24 h), and media and cell lysates were analyzed by HPLC-MS/MS to evaluate T1AM and TA1. To assess the endogenous production, cells were incubated with DMEM + 10%FBS or human serum without T1AM. To evaluate the presence of Ald, T1AM (100 µM) was incubated with bovine plasma amine oxidase (0.33 mg/ml). After protein precipitation (CAN?? ACN?), samples were centrifuged and supernatants analyzed by HPLC-MS/MS operating in Full Scan mode to detect ions of interest.

In NG108–15 and U87MG cells, in medium, T1AM at 0.1 and 1 µM rapidly decreased and became undetectable after 2–3 h. Differently, in the infusion with 10 µM, T1AM was measurable after 24 h. T1AM was taken up by cells and catabolized to TA1 which was detectable from the beginning of treatment (1 h), exceeding T1AM concentration after 24h. In cells incubated only with medium, neither T1AM nor TA1 were detected, in this way excluding any endogenous production by neuronal cells.

Putative aldehyde ion (m/z 353) was detected in negative ion mode without the formation of TA1. The fragmentation of this ion led to iodine loss, which reinforced the hypothesis that the detected molecule was a T1AM derivative.

In conclusion T1AM was taken up by cells and catabolized to TA1. The detection of the putative intermediate 3-iodothyroacetic aldehyde still needs to be confirmed by further studies.