Metabolomics of Small Intestine Neuroendocrine Tumors and Related Hepatic Metastases.

To assess the metabolomic fingerprint of small intestine neuroendocrine tumors (SI-NETs) and related hepatic metastases, and to investigate the influence of the hepatic environment on SI-NETs metabolome. Ninety-four tissue samples, including 46 SI-NETs, 18 hepatic NET metastases and 30 normal SI and liver samples, were analyzed using 1H-magic angle spinning (HRMAS) NMR nuclear magnetic resonance (NMR) spectroscopy. Twenty-seven metabolites were identified and quantified. Differences between primary NETs vs. normal SI and primary NETs vs. hepatic metastases, were assessed. Network analysis was performed according to several clinical and pathological features. Succinate, glutathion, taurine, myoinositol and glycerophosphocholine characterized NETs. Normal SI specimens showed higher levels of alanine, creatine, ethanolamine and aspartate. PLS-DA revealed a continuum-like distribution among normal SI, G1-SI-NETs and G2-SI-NETs. The G2-SI-NET distribution was closer and clearly separated from normal SI tissue. Lower concentration of glucose, serine and glycine, and increased levels of choline-containing compounds, taurine, lactate and alanine, were found in SI-NETs with more aggressive tumors. Higher abundance of acetate, succinate, choline, phosphocholine, taurine, lactate and aspartate discriminated liver metastases from normal hepatic parenchyma. Higher levels of alanine, ethanolamine, glycerophosphocholine and glucose was found in hepatic metastases than in primary SI-NETs. The present work gives for the first time a snapshot of the metabolomic characteristics of SI-NETs, suggesting the existence of complex metabolic reality, maybe characteristic of different tumor evolution.

Impact of real-time metabolomics in liver transplantation: Graft evaluation and donor-recipient matching.

BACKGROUND & AIMS: There is an emerging need to assess the metabolic state of liver allografts especially in the novel setting of machine perfusion preservation and donor in cardiac death (DCD) grafts. High-resolution magic-angle-spinning nuclear magnetic resonance (HR-MAS-NMR) could be a useful tool in this setting as it can extemporaneously provide untargeted metabolic profiling. The purpose of this study was to evaluate the potential value of HR-MAS-NMR metabolomic analysis of back-table biopsies for the prediction of early allograft dysfunction (EAD) and donor-recipient matching. METHOD: The metabolic profiles of back-table biopsies obtained by HR-MAS-NMR, were compared according to the presence of EAD using partial least squares discriminant analysis. Network analysis was used to identify metabolites which changed significantly. The profiles were compared to native livers to identify metabolites for donor-recipient matching. RESULTS: The metabolic profiles were significantly different in grafts that caused EAD compared to those that did not. The constructed model can be used to predict the graft outcome with excellent accuracy. The metabolites showing the most significant differences were lactate level >8.3 mmol/g and phosphocholine content >0.646 mmol/g, which were significantly associated with graft dysfunction with an excellent accuracy (AUROClactates = 0.906; AUROCphosphocholine = 0.816). Native livers from patients with sarcopenia had low lactate and glycerophosphocholine content. In patients with sarcopenia, the risk of EAD was significantly higher when transplanting a graft with a high-risk graft metabolic score. CONCLUSION: This study underlines the cost of metabolic adaptation, identifying lactate and choline-derived metabolites as predictors of poor graft function in both native livers and liver grafts. HR-MAS-NMR seems a valid technique to evaluate graft quality and the consequences of cold ischemia on the graft. It could be used to assess the efficiency of graft resuscitation on machine perfusion in future studies. LAY SUMMARY: Real-time metabolomic profiles of human grafts during back-table can accurately predict graft dysfunction. High lactate and phosphocholine content are highly predictive of graft dysfunction whereas low lactate and phosphocholine content characterize patients with sarcopenia. In these patients, the cost of metabolic adaptation may explain the poor outcomes.

Paraganglioma of the organ of Zuckerkandl associated with a somatic HIF2α mutation: A case report.

Paragangliomas of the organ of Zuckerkandl (OZ-PGL) are rare tumors that, in >70% of cases, occur in association with succinate dehydrogenase complex iron sulfur subunit B (SDHB) or SDHD gene mutations. The aim of the current study was to determine whether a somatic genetic defect in the hypoxia-inducible factor 2α (HIF2α) gene was present in a case of sporadic OZ-PGL. A 32-year-old African female presented with uncontrolled hypertension during the first trimester of pregnancy. A diagnostic hysteroscopy was performed 3 months after delivery, precipitating a hypertensive crisis. Thereafter, the patient was diagnosed with noradrenaline-secreting OZ-PGL. A complete blood count identified mild normocytic anemia of an inflammatory origin. Surgical removal of the tumor resulted in normalization of plasma and urinary normetanephrine levels. Genetic testing for germline mutations (including large deletions) in the von Hippel-Lindau tumor suppressor, SDHB, SDHC and SDHD genes was normal. However, a heterozygous missense mutation (c.1589Cys>Tyr) was detected in exon 12 of HIF2α, which results in a substitution of alanine 530 with valine (Ala530Val) in the HIF2α protein. A germline mutation was excluded based on the negative results of blood DNA testing. A three-dimensional homology model of Ala530Val was constructed, which showed impaired HIF2α/VHL interaction and decreased HIF2α ubiquitination. 1H-high-resolution magic-angle-spinning nuclear magnetic resonance spectroscopy detected low succinate levels and high α and ß glucose levels. To the best of our knowledge, the present case represents the first of its kind to associate a somatic HIF2α gain-of-function mutation with OZ-PGL. It is therefore recommended that patients without germline SDHx mutations should be tested for HIF2α mutations.

Identifying mitotane-induced mitochondria-associated membranes dysfunctions: metabolomic and lipidomic approaches.

Mitotane (o,p'DDD), the most effective drug in adrenocortical carcinoma, concentrates into the mitochondria and impacts mitochondrial functions. To address the molecular mechanisms of mitotane action and to identify its potential target, metabolomic and lipidomic approaches as well as imaging analyses were employed in human adrenocortical H295R cells allowing identification of Mitochondria-Associated Membranes dysfunction as a critical impact of mitotane. Study of intracellular energetic metabolites by NMR spectroscopy showed that mitotane significantly decreased aspartate while concomitantly increased glutamate content in a time- and concentration-dependent manner. Such alterations were very likely linked to the previously described, mitotane-induced respiratory chain defect. Lipidomic studies of intracellular and intramitochondrial phospholipids revealed that mitotane exposure markedly reduced the phosphatidylserine/phosphatidylethanolamine ratio, indicative of a dysfunction of phosphatidylserine decarboxylase located in Mitochondria-Associated Membranes. Expression levels of Mitochondria-Associated Membranes proteins phosphatidylserine decarboxylase, DRP1, ATAD3A or TSPO were greatly reduced by mitotane as assessed by western blot analyses. Mitotane exposure markedly altered endogenous Mitochondria-Associated Membranes integrity and reduced the magnitude of mitochondria and the endoplasmic reticulum interactions as demonstrated by high resolution deconvolution microscopy and quantification. Finally, we showed that PK11195, a pharmacological inhibitor of the cholesterol translocator TSPO, embedded in Mitochondria-Associated Membranes, exerts a synergetic effect with mitotane in inducing Mitochondria-Associated Membranes disruption, apoptosis and in inhibiting steroid secretion. Altogether, our results demonstrate Mitochondria-Associated Membranes dysfunction in H295R cells treated with mitotane and that TSPO inhibition significantly potentiates mitotane antitumoral and antisecretory actions in vitro. This constitutes a potential and promising pharmacological strategy for patients with adrenocortical carcinoma.

High-resolution magic angle spinning (1)H nuclear magnetic resonance spectroscopy metabolomics of hyperfunctioning parathyroid glands.

BACKGROUND: Primary hyperparathyroidism (PHPT) may be related to a single gland disease or multiglandular disease, which requires specific treatments. At present, an operation is the only curative treatment for PHPT. Currently, there are no biomarkers available to identify these 2 entities (single vs. multiple gland disease). The aims of the present study were to compare (1) the tissue metabolomics profiles between PHPT and renal hyperparathyroidism (secondary and tertiary) and (2) single gland disease with multiglandular disease in PHPT using metabolomics analysis. METHODS: The method used was (1)H high-resolution magic angle spinning nuclear magnetic resonance spectroscopy. Forty-three samples from 32 patients suffering from hyperparathyroidism were included in this study. RESULTS: Significant differences in the metabolomics profile were assessed according to PHPT and renal hyperparathyroidism. A bicomponent orthogonal partial least square-discriminant analysis showed a clear distinction between PHPT and renal hyperparathyroidism (R(2)Y = 0.85, Q(2) = 0.63). Interestingly, the model also distinguished single gland disease from multiglandular disease (R(2)Y = 0.96, Q(2) = 0.55). A network analysis was also performed using the Algorithm to Determine Expected Metabolite Level Alterations Using Mutual Information (ADEMA). Single gland disease was accurately predicted by ADEMA and was associated with higher levels of phosphorylcholine, choline, glycerophosphocholine, fumarate, succinate, lactate, glucose, glutamine, and ascorbate compared with multiglandular disease. CONCLUSION: This study shows for the first time that (1)H high-resolution magic angle spinning nuclear magnetic resonance spectroscopy is a reliable and fast technique to distinguish single gland disease from multiglandular disease in patients with PHPT. The potential use of this method as an intraoperative tool requires specific further studies.

Determination of the unmetabolised (18)F-FDG fraction by using an extension of simplified kinetic analysis method: clinical evaluation in paragangliomas.

Tumours with high (18)F-FDG uptake values on static late PET images do not always exhibit high proliferation indices. These discrepancies might be related to high proportion of unmetabolised (18)F-FDG components in the tissues. We propose a method that enables to calculate different (18)F-FDG kinetic parameters based on a new mathematical approach that integrates a measurement error model. Six patients with diagnosed non-metastatic paragangliomas (PGLs) and six control patients with different types of lesions were investigated in this pilot study using (18)F-FDG PET/CT. In all cases, a whole-body acquisition was followed by four static acquisitions centred over the target lesions, associated with venous blood samplings. We used an extension of the Hunter's method to calculate the net influx rate constant (K H). The exact net influx rate constant and vascular volume fraction (K i and V, respectively) were subsequently obtained by the method of least squares. Next, we calculated the mean percentages of metabolised (PM) and unmetabolised (PUM) (18)F-FDG components, and the times required to reach 80 % of the amount of metabolised (18)F-FDG (T80%). A test-retest evaluation indicated that the repeatability of our approach was accurate; the coefficients of variation were below 2 % regardless of the kinetic parameters considered. We observed that the PGLs were characterised by high dispersions of the maximum standardised uptake value SUVmax (9.7 ± 11, coefficient of variation CV = 114 %), K i (0.0137 ± 0.0119, CV = 87 %), and V (0.292 ± 0.306, CV = 105 %) values. The PGLs were associated with higher PUM (p = 0.02) and T80% (p = 0.02) values and lower k 3 (p = 0.02) values compared to the malignant lesions despite the similar SUVmax values (p = 0.55). The estimations of these new kinetic parameters are more accurate than SUVmax or K i for in vivo metabolic assessment of PGLs at the molecular level.

Metabolome profiling by HRMAS NMR spectroscopy of pheochromocytomas and paragangliomas detects SDH deficiency: clinical and pathophysiological implications.

Succinate dehydrogenase gene (SDHx) mutations increase susceptibility to develop pheochromocytomas/paragangliomas (PHEOs/PGLs). In the present study, we evaluate the performance and clinical applications of (1)H high-resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR) spectroscopy-based global metabolomic profiling in a large series of PHEOs/PGLs of different genetic backgrounds. Eighty-seven PHEOs/PGLs (48 sporadic/23 SDHx/7 von Hippel-Lindau/5 REarranged during Transfection/3 neurofibromatosis type 1/1 hypoxia-inducible factor 2α), one SDHD variant of unknown significance, and two Carney triad (CTr)-related tumors were analyzed by HRMAS-NMR spectroscopy. Compared to sporadic, SDHx-related PHEOs/PGLs exhibit a specific metabolic signature characterized by increased levels of succinate (P < .0001), methionine (P = .002), glutamine (P = .002), and myoinositol (P < .0007) and decreased levels of glutamate (P < .0007), regardless of their location and catecholamine levels. Uniquely, ATP/ascorbate/glutathione was found to be associated with the secretory phenotype of PHEOs/PGLs, regardless of their genotype (P < .0007). The use of succinate as a single screening test retained excellent accuracy in distinguishing SDHx versus non-SDHx-related tumors (sensitivity/specificity: 100/100%). Moreover, the quantification of succinate could be considered a diagnostic alternative for assessing SDHx-related mutations of unknown pathogenicity. We were also able, for the first time, to uncover an SDH-like pattern in the two CTr-related PGLs. The present study demonstrates that HRMAS-NMR provides important information for SDHx-related PHEO/PGL characterization. Besides the high succinate-low glutamate hallmark, SDHx tumors also exhibit high values of methionine, a finding consistent with the hypermethylation pattern of these tumors. We also found important levels of glutamine, suggesting that glutamine metabolism might be involved in the pathogenesis of SDHx-related PHEOs/PGLs.