Supplementary Material for: Urine Metabolomics by <sup>1</sup>H-NMR Spectroscopy Indicates Associations between Serum 3,5-T<sub>2</sub> Concentrations and Intermediary Metabolism in Euthyroid Humans M.Pietzner G.Homuth K.Budde I.Lehmphul U.Völker H.Völzke M.Nauck J.Köhrle N.Friedrich 2015 <b><i>Context:</i></b> 3,5-Diiodo-L-thyronine (3,5-T<sub>2</sub>) is a thyroid hormone metabolite which exhibited versatile effects in rodent models, including the prevention of insulin resistance or hepatic steatosis typically forced by a high-fat diet. With respect to euthyroid humans, we recently observed a putative link between serum 3,5-T<sub>2</sub> and glucose but not lipid metabolism. <b><i>Objective:</i></b> The aim of the present study was to widely screen the urine metabolome for associations with serum 3,5-T<sub>2</sub> concentrations in healthy individuals. <b><i>Study Design and Methods:</i></b> Urine metabolites of 715 euthyroid participants of the population-based Study of Health in Pomerania (SHIP-TREND) were analyzed by <sup>1</sup>H-NMR spectroscopy. Multinomial logistic and multivariate linear regression models were used to detect associations between urine metabolites and serum 3,5-T<sub>2</sub> concentrations. <b><i>Results:</i></b> Serum 3,5-T<sub>2</sub> concentrations were positively associated with urinary levels of trigonelline, pyroglutamate, acetone and hippurate. In detail, the odds for intermediate or suppressed serum 3,5-T<sub>2</sub> concentrations doubled owing to a 1-standard deviation (SD) decrease in urine trigonelline levels, or increased by 29-50% in relation to a 1-SD decrease in urine pyroglutamate, acetone and hippurate levels. <b><i>Conclusion:</i></b> Our findings in humans confirmed the metabolic effects of circulating 3,5-T<sub>2</sub> on glucose and lipid metabolism, oxidative stress and enhanced drug metabolism as postulated before based on interventional pharmacological studies in rodents. Of note, 3,5-T<sub>2</sub> exhibited a unique urinary metabolic profile distinct from previously published results for the classical thyroid hormones.