An Online Solid-Phase Extraction–Liquid Chromatography–Tandem

327 An online solid-phase extraction–liquid chromatography–tandem mass spectrometry method to study the presence of thyronamines in plasma 13 and tissue and their putative conversion from C6-thyroxine M T Ackermans1, L P Klieverik2, P Ringeling3, E Endert1, A Kalsbeek2,4 and E Fliers2 1Laboratory of Endocrinology, F2-131.1., Department of Clinical Chemistry and 2Department of Endocrinology and Metabolism, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands 3Spark Holland, P. de Keyserstraat 8, 7825 VE, Emmen, The Netherlands 4Netherlands Institute for Neuroscience, Meibergdreef 47, 1105 BA, Amsterdam, The Netherlands (Correspondence should be addressed to M T Ackermans; Email: [email protected]) Abstract 13 Thyronamines are exciting new players at the crossroads of or brain samples of rats treated with C6-T4. Surprisingly, thyroidology and metabolism. Here, we report the develop- our method did not detect any endogenous T1AM or T0AM ment of a method to measure 3-iodothyronamine (T1AM) in plasma from vehicle-treated rats, nor in human plasma or and thyronamine (T0AM) in plasma and tissue samples. thyroid tissue. Although we are cautious to draw general The detection limit of the method was 0.25 nmol/l in plasma conclusions from these negative findings and in spite of the and 0.30 pmol/g in tissue both for T1AM and for T0AM. fact that insufficient sensitivity of the method related to Using this method, we were able to demonstrate T1AM and extractability and stability of T0AM cannot be completely T0AM in plasma and liver from rats treated with synthetic excluded at this point, our findings raise questions on the thyronamines. Although we demonstrated the in vivo biosynthetic pathways and concentrations of endogenous 13 13 13 conversion of C6-thyroxine ( C6-T4)to C6-3,5, T1AM and T0AM. 0 13 3 -triiodothyronine, we did not detect C6-T1AM in plasma Journal of Endocrinology (2010) 206, 327–334 Introduction (Klieverik et al. 2009). These findings prompted us to develop an online solid-phase extraction (SPE)–liquid chromato- Thyronamines are structural homologs of thyroid hormones graphy (LC)–tandem mass spectrometry method (XLC–MS) (see Fig. 1). In the early 1970s, Dr M Dratman et al.(Dratman to study the bioavailability of thyronamines in plasma and 1974) speculated about their putative biosynthesis and action. tissues including the brain and the thyroid gland, as well as to Cody et al. (1984) described the molecular structure and study their hypothesized formation from thyroxine (T4) using 0 13 biochemical activity of 3,5,3 -triiodothyronamine (T3AM) in stably labeled C6-T4. 1984. It was, however, not until 2004 that Scanlan et al. (2004) showed a major physiological role for thyronamines based upon elegant experiments in rodents. Systemic administration Materials and Methods of 3-iodothyronamine (T1AM) and, to a lesser extent, of Plasma and tissue samples thyronamine (T0AM) induces profound metabolic and cardiac effects including bradycardia, hypothermia, and We used heparinized plasma samples of thyronamine-treated hyperglycemia (Scanlan et al. 2004, Braulke et al. 2008, (i.p. bolus of either 50 mg/kg T1AM or 50 mg/kg T0AM in Zucchi et al. 2008, Dhillo et al. 2009, Klieverik et al. 2009). 500 ml) or vehicle-treated (i.p. bolus of 500 ml saline) euthyroid, Both in vitro and in vivo studies have proposed potential adult Wistar rats (nZ6) that participated in the studies published receptors for thyronamines, i.e. the trace amine-associated previously (Klieverik et al.2008, 2009). Moreover, plasma, receptor 1 (Hart et al. 2006, Grandy 2007, Tan et al. 2009, hypothalamus, and neocortex samples of rats (nZ2 per dose) 13 Panas et al. 2010) and adrenergic receptor a2(Regard et al. treated with C6-T4 were analyzed to investigate whether 2007). Furthermore, studies have been published addressing T1AM is formed from T4. These rats had been equipped with a 13 intra-cellular transport (Ianculescu et al. 2009) and meta- s.c. osmotic minipump delivering C6-T4 for a period of bolism (Piehl et al. 2008b, Wood et al. 2009) of thyronamines. 10 days (Alzet 2ml2, Durect Corp., Cupertino, CA, USA; flow We recently reported that both T1AM and T0AM can act in rate 5 ml/h; dose: vehicle, 0.44, 1.75, and 20 mg/100 g body the central nervous system to modulate glucose metabolism weight per day; Klieverik et al. 2008). Furthermore, we studied Journal of Endocrinology (2010) 206, 327–334 DOI: 10.1677/JOE-10-0060 0022–0795/10/0206–327 q 2010 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/27/2021 05:08:20PM via free access 328 M T ACKERMANS and others . XLC–MS of thyronamines in plasma and tissue I I CO2H T0AM in PBS were added to 100 ml plasma. Plasma was vortexed and processed as unspiked plasma. For the thyroid NH2 tissue samples, the same amount of T1AM and T0AM was HO O added to 100 mg tissue at the same time as the internal standard Thyroxine, T4 (IS). The recovery was calculated as the concentration of I I thyronamines measured divided by the concentration added. Sample pretreatment NH2 HO O All quantitative analyses were carried out using the IS method using stable isotope-labeled T1AM. As IS, d4-T1AM (1 mMin 3-Iodothyronamine, T1AM PBS) was used. In this molecule, four hydrogen atoms are I replaced by four deuterium atoms. As hydrogen and deuterium are isotopes of the same element, d4-T1AM and NH2 T1AM act similarly both biologically and analytically. HO O However, due to the higher atomic mass of deuterium compared with hydrogen, the molecular weight of d4-T1AM Thyronamine, T0AM is 4 mass units higher than that of T1AM enabling the distinction between the two molecules by mass spectrometry. Figure 1 Structural formulae of T4,T1AM, and T0AM. No d4-T1AM was added to the samples that were used to 13 13 human heparin plasma samples and serum (nZ8) taken from study the conversion from C6-T4 to C6-T1AM. healthy volunteers by venipuncture. Finally, we studied human thyroid tissue specimens (nZ2) obtained from the thyroid tissue Plasma samples Ten microliters IS were added to 100 ml bank in the Academic Medical Center of the University of plasma. Samples were incubated overnight (15–17 h) at 37 8C Amsterdam. As a positive control for the extraction of with 20 ml proteinase K (100 mg/ml in distilled water). thyronamines from tissue, we used (nZ2) liver samples from Thereafter, the sample was centrifuged (3 min at 16 000 g). the T1AM- and T0AM-treated rats. All the plasma and tissue Five microliters of the supernatant were diluted 20 times with samples were frozen immediately and thawed only once. All distilled water. This dilution was used for the measurement of experiments were performed in accordance with the guidelines protein fragments using SDS-PAGE and Coomassie Brilliant of either the Medical Ethics Committee of the Academic Blue coloring. Toanother 100 ml of the supernatant, 150 mlof Medical Center in Amsterdam or the Animal Care Committee 0.1% formic acid were added. The sample was placed at 4 8C of the Royal Netherlands Academy of Arts and Sciences. until analysis with XLC–MS. Tissue samples Ten microliters IS and 3 ml prechilled Chemicals KH2PO4 solution (100 mM at pH 6) were added to ca. Stock solutions of 10 mM T0AM, T1AM, d4-T1AM, and 100 mg tissue. The tissue was ground until it was devoid of 13 C6-T1AM in dimethyl sulfoxide (DMSO) were kindly visible tissue fragments. Six milliliters of chilled acetic acetone supplied by Dr T S Scanlan and Dr D K Grandy (Oregon (5 ml of 37% HCl in 100 ml acetone) were added. In order to Health and Science University, Portland, USA). Proteinase K, complete denaturation, the sample was placed on ice for recombinant, PCR grade, and PBS, SDS, and TRIS were 10 min. After centrifugation (10 min at 4000 g), the obtained from Roche. Coomassie Brilliant Blue G-250, supernatant was transferred to a clean tube and evaporated ammonium persulfate, tetramethylethylenediamine, acryla- to dryness followed by the addition of 300 mlof0.1% formic mide/bisacrylamide, and the Protein Plus Marker were acid. The supernatant obtained after centrifugation (5 min at obtained from Bio-Rad. ApoA1/ApoB calibrator was 4000 g) was placed at 4 8C until XLC–MS analysis. obtained form Abbott Diagnostics (Abbott Park). All other chemicals were obtained from Merck. Calibration solutions Serial dilutions of the stock solutions of T1AM and T0AM (10 mM in DMSO) at 1, 5, 10, 20, 30, 40, and 50 nmol/l were prepared in 0.1% formic Recovery experiments acid. For XLC–MS, 100 ml of each calibration standard were The recovery of the SPE was tested by comparing peak areas of pipetted in a deep well plate. Toeach well, 10 ml IS and 140 ml identical injections of T1AM, T0AM, and d4-T1AM with and 0.1% formic acid were added. Before the deep well plate without SPE (nZ10). In order to test the recovery of the was put into the autosampler it was covered, vortexed, and sample pretreatment including the stability of T1AM and centrifuged (2 min at 1000 g) to make sure that all the samples T0AM during the procedure, we processed human plasma and were well mixed, and that no air bubbles were left in the wells. human thyroid after adding T1AM and T0AM to the sample. For the linearity check, calibration was extended with For the plasma samples, 10 ml of a mix of 1 mmol/l T1AM and concentrations of 100 and 200 nmol/l T1AM and T0AM.

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