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J C E M B r i e f R e p o r t — E n d o c r i n e O N L I N E R e s e a r c h Detection of 3-Iodothyronamine in Human Patients: A Preliminary Study Elena Galli,* Maja Marchini,* Alessandro Saba, Sergio Berti, Massimo Tonacchera, Paolo Vitti, Thomas S. Scanlan, Giorgio Iervasi, and Riccardo Zucchi Scuola Superiore S. Anna (E.G.), 56127 Pisa, Italy; Dipartimento di Scienze dell’Uomo e dell’Ambiente (M.M., A.S., R.Z.) and Dipartimento di Endocrinologia (M.T., P.V.), University of Pisa, 56126 Pisa, Italy; Fondazione Gabriele Monasterio (S.B.), Ospedale del Cuore, 54100 Massa, Italy; Departments of Physiology and Pharmacology and Cell and Developmental Biology (T.S.S.), Oregon Health and Science University, Portland, Oregon 97239; and Istituto di Fisiologia Clinica (G.I.), 56124 Pisa, Italy Context and Objective: The primary purpose of this study was to detect and quantify 3-iodothyronamine (T1AM), an endogenous biogenic amine related to thyroid hormone, in human blood. Design: T1AM, total T3, and total T4 were assayed in serum by a novel HPLC tandem mass spectrometry assay, which has already been validated in animal investigations, and the results were related to standard clinical and laboratory variables. Setting and Patients: The series included one healthy volunteer, 24 patients admitted to a cardiological ward, and 17 ambulatory patients suspected of thyroid disease, who underwent blood sampling at admission for routine diagnostic purposes. Seven patients were affected by type 2 diabetes, and six patients showed echocardiographic evidence of impaired left ventricular function. Interventions: No intervention or any patient selection was performed. Main Outcome Measures: serum T1AM, total and free T3 and T4, routine chemistry, routine hematology, and echocardiographic parameters were measured. Results: T1AM was detected in all samples, and its concentration averaged 0.219 ⫾ 0.012 pmol/ml. The T1AM concentration was significantly correlated to total T4 (r ⫽ 0.654, P ⬍ 0.001), total T3 (r ⫽ 0.705, P ⬍ 0.001), glycated hemoglobin (r ⫽ 0.508, P ⫽ 0.013), brain natriuretic peptide (r ⫽ 0.543, P ⫽ 0.016), and ␥-glutamyl transpeptidase (r ⫽ 0.675, P ⬍ 0.001). In diabetic vs. nondiabetic patients T1AM concentration was significantly increased (0.232 ⫾ 0.014 vs. 0.203 ⫾ 0.006 pmol/ml, P ⫽ 0.044), whereas no significant difference was observed in patients with cardiac dysfunction. Conclusions: T1AM is an endogenous messenger that can be assayed in human blood. Our results are consistent with the hypothesis that circulating T1AM is produced from thyroid hormones and encourage further investigations on the potential role of T1AM in insulin resistance and heart failure. (J Clin Endocrinol Metab 97: E69 –E74, 2012) -Iodothyronamine (T1AM) is a decarboxylated and partially deiodinated derivative of thyroid hormone, which can interact with different molecular targets, namely (1– 8) trace amine associated receptor 1, and possibly other 3 members of the same family of G protein-coupled receptors; several monoamine transporters and organic anion transporters, including thyroid hormone transporters; mitochondrial F0F1-ATP synthase. Exogenous T1AM has been re- ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2012 by The Endocrine Society doi: 10.1210/jc.2011-1115 Received March 29, 2011. Accepted September 28, 2011. First Published Online October 25, 2011 * E.G. and M.M. contributed equally to this work. Abbreviations: BNP, Brain natriuretic peptide; FT3, free T3; FT4, free T4; ␥GT, ␥-glutamyl transpeptidase; HbA1c, glycated hemoglobin; HPLC-MS/MS, HPLC coupled to tandem mass spectrometry; T1AM, 3-iodothyronamine. J Clin Endocrinol Metab, January 2012, 97(1):E69 –E74 jcem.endojournals.org E69 E70 Galli et al. T1AM in Human Blood ported to produce a wide array of functional effects in isolated organs and whole animals, such as reduced body temperature (1), reduced heart rate and contractility (1, 9, 10), reduced glucose and increased lipid metabolism (11), decreased insulin and increased glucagon secretion (12, 13), and either increased (14) or decreased (15) food intake. T1AM has been detected in animal tissues by semiquantitative techniques (1, 9, 11), but the presence of T1AM in human blood has been disputed because after a few preliminary reports that were presented at scientific meetings (8, 16), negative results have been published (17). This is a crucial issue because detecting T1AM in human blood would be consistent with a role as a chemical messenger and provide the basis to investigate its role in human physiology and pathophysiology. We have recently developed a novel technique, based on HPLC coupled to tandem mass spectrometry (HPLC-MS/MS), which allowed quantitative assay of T1AM in rat blood and tissues (18). In the present investigation this method was used in a small series of human blood samples. The primary aim was to prove that T1AM is an endogenous compound in humans. In addition, we evaluated the relationship between T1AM concentration, thyroid hormone concentration, and some common clinical chemistry variables. Materials and Methods This investigation involved two groups of patients. The first group (group A) consisted of 24 consecutive patients admitted to the Cardiological Division of Massa Hospital (Massa, Italy). Because the results obtained in this group suggested a correlation between T1AM and thyroid hormone concentration, we also investigated 17 patients affected by or suspected of thyroid disease who were followed up by the cardiovascular risk unit of the Institute of Clinical Physiology in Pisa (group B). Blood samples were taken at admission, and HPLC-MS/MS assays were performed whenever a sufficient amount of blood was available after performing the routine laboratory examinations that were prescribed for diagnostic purposes. Free T3 (FT3) and free T4 (FT4) were determined by the Architect chemiluminescent microparticle immunoassay (Abbott Diagnostics, Abbott Park, IL). The final diagnosis was established on the basis of echocardiography, coronary arteriography, or peripheral angiography, as required. All patients gave informed consent to the present investigations, which was approved by the institutional ethical committee. A healthy volunteer (one of the authors of this paper) was also included in the study. Serum samples were collected (1 ml), placed in a 15 ml Teflon centrifuge tube and spiked with appropriate internal standards, i.e. 6 pmol D4-T1AM, 50 pmol 13C6-T4, or 50 pmol of 13C6-T3. Sample processing and an HPLC-MS/MS assay were performed as described previously (18). The T1AM assay was characterized by a limit of detection around 12.5 pg/ml (35 fmol/ml). Good linearity was observed within 3.5 orders of magnitude (r ⫽ 0.996), whereas intra- and interassay coefficients of variation were 7% or less and 12% or less, respectively (the lowest T1AM J Clin Endocrinol Metab, January 2012, 97(1):E69 –E74 concentration tested was 0.05 pmol/ml). The assays were performed on different days, and to reduce variability, serum samples from the healthy volunteer were included in each set and used as a calibration reference. Results are expressed as mean ⫾ S⌭⌴. Differences between groups were evaluated by unpaired t test. Correlations between variables were evaluated by linear regression analysis. The threshold of statistical significance was set at P ⫽ 0.05. GraphPad Prism version 4.1 for Windows (GraphPad Software, San Diego, CA) was used for data processing and statistical analysis. Results Our series included 20 males and 22 females, with an average age of 58.1 ⫾ 1.6 yr. In group A the reason for admission was chest pain in 12 patients, dyspnea in seven patients, intermittent claudication in two patients, repetitive transient ischemic attacks in one patient, syncope in one patient, and control of abdominal aortic aneurism in one patient. The final diagnosis was coronary artery disease in six patients, valvular heart disease in three patients, dilated cardiomyopathy in two patients, chronic atrial fibrillation in one patient, internal carotid artery stenosis in two patients, peripheral vascular disease with lower limb ischemia in two patients, and abdominal aortic aneurism in one patient. Seven patients admitted for chest pain were found to be free from significant coronary artery disease. Group B included four patients affected by untreated hypothyroidism due to autoimmune thyroiditis, three patients affected by untreated hyperthyroidism (Graves’ disease or multinodular toxic goiter), three athyreotic patients treated with exogenous levothyroxine yielding normal FT3 and FT4, and seven patients with normal thyroid function. Two group A patients were chronically taking oral L-thyroxine at suppressive dosage because of multinodular goiter. Most patients received additional chronic pharmacological treatment (see Table 1), which was not discontinued due to ethical reasons. The average values for routine laboratory examinations and echocardiographic variables are summarized in Table 1. Representative HPLC-MS/MS tracings are shown in Fig. 1 (panel A), and the overall results are included in Table 1. T1AM was detected in all patients and its serum concentration averaged 0.219 ⫾ 0.012 pmol/ml, whereas serum T3 and T4 concentrations averaged 1.52 ⫾ 0.11 and 142.32 ⫾ 16.20 pmol/ml, respectively. The ratio of FT3 to T3 was on the order of 0.2%, whereas the ratio of FT4 to T4 was on the order of 0.01%. These ratios are similar to the ratio of free vs. total hormone content as estimated by conventional techniques (18). To investigate the determinants of T1AM concentration, we analyzed its correlation with different clinical and J Clin Endocrinol Metab, January 2012, 97(1):E69 –E74 TABLE 1. jcem.endojournals.org E71 Clinical and laboratory variables Variable T1AM (pmol/ml) T3 (pmol/ml) T4 (pmol/ml) FT3 (fmol/ml) FT4 (fmol/ml) TSH (IU/ml) Age (yr) Weight (kg) Height (cm) BMI (kg/m2) BSA (m2) Hb (g/dl) Hct (%) Glucose (mg/dl) HbA1c (%) Creatinine (mg/dl) Urea (mg/dl) Uricemia (mg/dl) Albumin (g/dl) Proteins (g/dl) BNP (ng/ml) AST (IU/ml) ALT (IU/ml) ␥GT (IU/ml) ALP (IU/ml) CK (IU/ml) Fibrinogen (mg/dl) Cholesterol (mg/dl) HDL cholesterol (mg/dl) LDL cholesterol (mg/dl) Triglycerides (mg/dl) Heart rate (beats/min) Systolic AP (mm Hg) Diastolic AP (mm Hg) Left ventricle EDV (ml) Left ventricle EF (%) Left ventricle mass (g) Mean ⴞ SEM 0.219 ⫾ 0.012 1.52 ⫾ 0.11 142.32 ⫾ 16.20 3.18 ⫾ 0.26 13.46 ⫾ 0.96 6.65 ⫾ 2.88 58 ⫾ 2 78 ⫾ 4 169 ⫾ 2 27.4 ⫾ 1.3 1.89 ⫾ 0.04 13.1 ⫾ 0.4 38.9 ⫾ 1.7 119 ⫾ 7 6.0 ⫾ 0.2 1.00 ⫾ 0.06 44.1 ⫾ 4.1 5.2 ⫾ 0.3 3.7 ⫾ 0.1 6.7 ⫾ 0.1 122 ⫾ 26 18 ⫾ 1 16 ⫾ 2 31 ⫾ 5 67 ⫾ 5 104 ⫾ 12 297 ⫾ 14 175 ⫾ 6 54 ⫾ 2 101 ⫾ 6 97 ⫾ 6 70 ⫾ 3 129 ⫾ 4 75 ⫾ 2 127 ⫾ 9 54 ⫾ 2 225 ⫾ 11 Correlation coefficient vs. T1AM 1 0.705 0.654 0.550 0.339 ⫺0.049 0.013 0.152 0.183 0.050 0.172 0.216 0.270 0.198 0.508 ⫺0.079 ⫺0.029 0.023 0.020 ⫺0.135 0.543 0.138 0.008 0.675 0.121 0.194 0.337 ⫺0.186 ⫺0.039 ⫺0.227 0.172 0.173 ⫺0.148 ⫺0.249 0.051 ⫺0.082 0.084 P value ⬍0.001 ⬍0.001 ⬍0.001 0.037 NS NS NS NS NS NS NS NS NS 0.013 NS NS NS NS NS 0.016 NS NS ⬍0.001 NS NS 0.115 NS NS NS NS NS NS NS NS NS NS Data represent mean ⫾ SEM of 42 patients (group A ⫹ group B) with regard to T1AM, T3, T4, FT3, FT4, TSH, and age and 25 patients (group A) for the other variables. In some patients a few variables were not available: there were 10 missing values for left ventricle EDV, six missing values for BNP, five missing values for left ventricle mass, and three or fewer missing values in all other cases. Values of P ⱖ 0.200 are not shown. See text for correlations in specific subgroups. Several patients were receiving chronic pharmacological treatment. Assumed drugs included furosemide, thiazide diuretics, nitrates, etezimibe, proton pump inhibitors, digoxin, cetirizime, allopurinol, aliskiren, monoxidine, trimetazidine, low-molecularweight heparin, sulfonylureas, and metformin. Five patients were assuming levothyroxine, as detailed in Results. BMI, Body mass index; BSA, body surface area, Hb, hemoglobin; Hct, hematocrit; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alcaline phosphatase; CK, creatine kinase; HDL, high-density lipoprotein; LDL, low-density lipoprotein; AP, aortic pressure; EDV, end diastolic volume; EF, ejection fraction; NS, not significant. biochemical variables. The results are included in Table 1, whereas selected scatter plots are shown in Fig. 1 (panels B–E). T1AM concentration was significantly related to T4 (r ⫽ 0.654, P ⬍ 0.001), T3 (r ⫽ 0.705, P ⬍ 0.001), FT4 (r ⫽ 0.339, P ⫽ 0.037), and FT3 (r ⫽ 0.550, P ⬍ 0.001). A significant correlation with T4 was observed even if analysis was limited to group A patients (r ⫽ 0.450, P ⫽ 0.024). Significant correlations were also observed between T1AM and glycated hemoglobin (HbA1c; r ⫽ 0.508, P ⫽ 0.013), brain natriuretic peptide (BNP; r ⫽ 0.543, P ⫽ 0.016), or ␥-glutamyl transpeptidase (␥GT; r ⫽ 0.675, P ⬍ 0.001). Because HbA1c is widely used as an index of persistent hyperglycemia, we investigated whether diabetes may be associated with differences in T1AM levels. In group A, a full screening for diabetes was performed, and seven patients were diagnosed as affected by type 2 diabetes on the basis of standard clinical criteria, i.e. fasting blood glucose 126 mg/dl or greater and/or abnormal response to an oral glucose tolerance test. T1AM concentration was significantly higher in these patients than in the other, nondiabetic, group A patients (n ⫽ 18) (0.232 ⫾ 0.014 vs. 0.203 ⫾ 0.006 pmol/ml, P ⫽ 0.044), whereas no significant difference was observed for all the other variables, E72 Galli et al. T1AM in Human Blood J Clin Endocrinol Metab, January 2012, 97(1):E69 –E74 between T1AM concentration and BNP became very close (r ⫽ 0.925, P ⫽ 0.027). Discussion Exogenous T1AM produces a wide array of functional effects, which are often opposite those produced on a longer time scale by thyroid hormone (1–10). Because T1AM could be detected in animal tissues and blood, the concept that it is a novel chemical messenger developed, leading to speculations about its potential physiological and pathophysiological role. However, the presence of T1AM in human blood has been disputed (17). To clarify this crucial issue, we studied a small series of consecutive patients admitted to a cardiological ward, using a novel HPLC-MS/MS method, which has previously allowed T1AM detection in rat blood and tissue homogenates, as well as in a single huFIG. 1. The upper panel (A) shows a representative selected reaction monitoring man blood sample (16). chromatogram obtained in one patient. The peaks represent the following transitions: m/z T1AM was detected in all patients, 356.2 3 212.2 (T1AM, peak at 2.41 min, expanded 100-fold), 651.8 3 605.8 (T3, peak at 3.41 min, expanded 10-fold), and m/z 777.8 3 731.9 (T4, peak at 3.66 min). Peak identity and its concentration averaged 0.219 was confirmed by analyzing two additional transitions and by comparison with the pmol/ml, which is similar to the concorresponding internal standards (not shown in this figure). The lower panels include scatter centration that we have previously plots showing the correlation between T1AM and selected variables, namely T4 (n ⫽ 42) (B); measured in rat serum (18), and about HBA1c (n ⫽ 23) (C); BNP (n ⫽ 19) (D); and ␥GT (n ⫽ 23) (E). Please note that some laboratory data were not available in all patients. Statistical analysis was performed by linear regression 1 order of magnitude lower than serum and the results are shown in the panels. T3. With our method, greater than 90% recovery of exogenous T1AM, T3, or T4 except for fasting blood glucose (157 ⫾ 15 vs. 103 ⫾ 4 added to human blood samples or rat tissue homogenates mg/dl, P ⬍ 0.001) and glycated hemoglobin (7.08 ⫾ 0.35 was observed (18), and the values that we measured for T 3 vs. 5.42 ⫾ 0.07%, P ⬍ 0.001). and T4 are similar to the total T3 and T4 values, which are BNP is used as an endocrine marker of heart failure, and usually obtained by conventional techniques (19). Thereprogressive increase in BNP is considered as an index of fore, we believe that our HPLC-MS/MS assay provides a the progression of heart failure. In group A, six patients measurement of total T3 and T4 concentrations and specshowed evidence of cardiac dysfunction on the basis of an ulate that the same may happen for T1AM. echocardiographic left ventricular ejection fraction less However, it should be acknowledged that we are still than 50%. If cardiac dysfunction patients were compared unable to evaluate T1AM binding to proteins and to diswith the other group A patients (n ⫽ 19), the former showed higher values of heart rate (91 ⫾ 5 vs. 63 ⫾ 2 beats criminate free and bound T1AM. In a recent report, Hoefig per minute, P ⬍ 0.001), left ventricle end diastolic volume et al. (20) described a chemiluminescent immunoassay (183 ⫾ 14 vs. 103 ⫾ 6 ml, P ⬍ 0.001), left ventricle ejec- that confirmed the presence of T1AM in human blood but tion fraction (41.5 ⫾ 3.3 vs. 58.7 ⫾ 0.7%, P ⬍ 0.001), and provided estimated values on the order of 66 pmol/ml. left ventricle mass (264 ⫾ 23 vs. 207 ⫾ 11 g, P ⫽ 0.021), Further investigations will be required to understand the but no significant difference was observed for all the other reasons for this discrepancy. Immunological methods variables, including T1AM concentration (0.209 ⫾ 0.021 raise questions about the specificity of the antibody and its vs. 0.212 ⫾ 0.005 pmol/ml). However, within the pa- relative affinity for free vs. protein-bound antigens. In partients with reduced cardiac function, the correlation ticular, cross-reactivity might exist with endogenous J Clin Endocrinol Metab, January 2012, 97(1):E69 –E74 T1AM derivatives, such as sulfated T1AM, and serum might contain a mixture of T1AM and chemically modified T1AM, with equilibrium largely favoring modified T1AM (21). Mass spectrometry-based methods are usually considered as a gold standard for specificity, but quantification from biological matrices is critically dependent on the sample preparation steps, and we cannot exclude that a fraction of tightly-bound T1AM is not released by our extraction procedure. In this initial investigation, the available routine laboratory data were used to identify potential determinants of T1AM concentration. A significant correlation was observed between T1AM and T4, T3, FT4, or FT3. Although the Architect FT4 assay (Abbott Diagnostics) may not be completely reliable because its results have been shown not to correlate with logTSH (22), our findings are consistent with the hypothesis that T1AM is produced by decarboxylation and deiodination of T4 in peripheral tissues. In rats the highest levels of endogenous T1AM were detected in liver (18). Because increased plasma ␥GT is regarded as an index of hepatocyte injury and/or cholestasis, the correlation between T1AM and plasma ␥GT might reflect T1AM release from hepatocytes. T1AM concentration was significantly increased in diabetic patients and significantly correlated with HbA1c levels. Exogenous T1AM administration has been reported to stimulate fatty acid oxidation vs. glucose oxidation (11) and to favor glucagon secretion over insulin secretion (12, 13). These observations suggest that endogenous T1AM might play a role in insulin resistance and in type 2 diabetes. Our series is too small to draw further conclusions, although the existence of a positive correlation between T1AM and BNP suggests that the relationship between T1AM and heart failure deserves further attention, even if no significant change in T1AM concentration occurred in patients with impaired left ventricular function. In conclusion, T1AM is an endogenous substance that can be consistently assayed in human blood. This pilot investigation is consistent with peripheral production of T1AM from thyroid hormone and suggests a potential role of T1AM in insulin resistance. Acknowledgments Address all correspondence and requests for reprints to: R. Zucchi, M.D., Dipartmento di Scienze dell’Uomo e dell’Ambiente, via Roma 55, 56126 Pisa, Italy. E-mail: [email protected]. This work was supported by the Ministero dell’Università e della Ricerca, Italy (Cofin 2008, to R.Z.) and by the AmbiSEN center. jcem.endojournals.org E73 Disclosure Summary: There are no conflict of interest to disclose for any author. References 1. Scanlan TS, Suchland KL, Hart ME, Chiellini G, Huang Y, Kruzich PJ, Frascarelli S, Crossley DA, Bunzow JR, Ronca-Testoni S, Lin ET, Hatton D, Zucchi R, Grandy DK 2004 3-Iodothyronamine is an endogenous and rapid-acting derivative of thyroid hormone. Nat Med 10:638 – 642 2. Snead AN, Santos MS, Seal RP, Miyakawa M, Edwards RH, Scanlan TS 2007 Thyronamines inhibit plasma membrane and vescicular monoamine transport. ACS Chem Biol 2:390 –398 3. Frascarelli S, Ghelardoni S, Chiellini G, Vargiu R, Ronca-Testoni S, Scanlan TS, Grandy DK, Zucchi R 2008 Cardiac effects of trace amines: pharmacological characterization of trace amine associated receptors. Eur J Pharmacol 587:231–236 4. 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