Supplementary Figure 1 Examples of 1H NMR spectra for different genotypes A. Example of NMR spectrum of a sporadic tumor depicting the peak positions of lactic acid, acetic acid and catecholamines. B. Example of NMR spectrum of a SDHB tumor depicting peak position of succinic acid. C. Example of NMR spectrum of a RET tumor depicting ATP/ADP/AMP and adrenaline specific peaks. Supplementary Figure 2 Comparison of epinephrine and norepinephrine content in tumor tissues as determined by HPLC and 1H-NMR spectroscopy. Passing - Bablok regression plot to compare measurement of epinephrine and norepinephrine content in PGL tumor tissues by HPLC and 1HNMR spectroscopy expressed as μg per mg tissue in Log10 scale. Supplementary Figure 3: Activities of respiratory chain complex II and tumor tissue succinate content in SDH related tumors. Black bars represent complex II activity expressed in mU/mg protein and grey bars represent tumor tissue succinate content in mmol/mg tissue for each individual tumor. Represented on X axis is the hereditary mutation in the patient from whom the tumor was resected.
ARTICLE ABSTRACT
Purpose: Pheochromocytomas and paragangliomas (PGL) are neuroendocrine tumors of sympathetic and parasympathetic paraganglia. This study investigated the relationships between genotype-specific differences in mitochondrial function and catecholamine content in PGL tumors.Experimental Design: Respiratory chain enzyme assays and 1H-nuclear magnetic resonance (NMR) spectroscopy at 500 MHz were conducted on homogenates of 35 sporadic PGLs and 59 PGLs from patients with hereditary mutations in succinate dehydrogenase subunits B and D (SDHB, SDHD), succinate dehydrogenase assembly factor 2, von Hippel-Lindau (VHL), rearranged during transfection (RET), neurofibromatosis type 1 (NF1), and myc-associated factor X.Results: In SDHx-related PGLs, a significant decrease in complex II activity (P < 0.0001) and a significant increase in complex I, III, and IV enzyme activities were observed when compared to sporadic, RET, and NF1 tumors. Also, a significant increase in citrate synthase (P < 0.0001) enzyme activity was observed in SDHx-related PGLs when compared to sporadic-, VHL-, RET-, and NF1-related tumors. An increase in succinate accumulation (P < 0.001) and decrease in ATP/ADP/AMP accumulation (P < 0.001) was observed when compared to sporadic PGLs and PGLs of other genotypes. Positive correlations (P < 0.01) were observed between respiratory chain complex II activity and total catecholamine content and ATP/ADP/AMP and total catecholamine contents in tumor tissues.Conclusions: This study for the first time establishes a relationship between determinants of energy metabolism, like activity of respiratory chain enzyme complex II, ATP/ADP/AMP content, and catecholamine content in PGL tumors. Also, this study for the first time successfully uses NMR spectroscopy to detect catecholamines in PGL tumors and provides ex vivo evidence for the accumulation of succinate in PGL tumors with an SDHx mutation. Clin Cancer Res; 19(14); 3787–95. ©2013 AACR.
