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Hintergrund und ZielMikrobiologische Alkylierungsraten sind im Hinblick auf den unter naturlichen Bedingungen stattfindenden Ruckgang der Selenkonzentrationen bzw. -frachten kontaminierter Okosysteme von Interesse. Material und MethodenZur Analyse der leichtfluchtigen Organoselenverbindungen im Deponiegas wie auch uber die mikrobiologischen Inkubationsansatze wurde die Gasphase in Tedlar®-Beuteln gesammelt. Die Kopplung von effizienter Anreicherung (cryotrapping-cryofocusing), gaschromatografischer Trennung und einem relativ nachweisstarken elementselektiven Detektor als CT-CF-GC-ICP-MS ermoglichte eine direkte Selenspeziation in der Gasphase mit Nachweisgrenzen im Ultraspurenbereich (pg Se) sowohl im Deponiegas als auch in den Kulturansatzen. ErgebnisseDie Inkubationsansatze mit Alternaria alternata als aktiv methylierender Organismus zeigten uber den gesamten Konzentrationsbereich (10 µg Se-IV L–1 bis 10 mg Se-IV L–1) eine aktive Produktion verschiedener leichtfluchtiger Organoselenverbindungen (DMSe, DMDSe, EMDSe, DEDSe) in der Grosenordnung von bis zu 10 mg m–3 d–1 in der Gasphase. Bis zu 7 % der Selenkonzentration von 1 mg L–1 im Nahrmedium wurden so binnen einer Woche verfluchtigt. Diese volatilen Selenspezies wurden auch im Deponiegas beobachtet DiskussionIm Vergleich zu anderen Studien mit mikrobiologischen Reinkulturen sowie Isolaten aus Wassern und Boden sind die Ausbeuten an volatilen Organoselenverbindungen in den Inkubationsansatzen mit A. alternata und dem Deponiesickerwasser ahnlich hoch, wobei der hier untersuchte Konzentrationsbereich allerdings weit unterhalb des in der Literatur fur Inkubationsansatze bisher berichteten lag. SchlussfolgerungenDa die Alkylierung von Selen durch A. alternata in Mischkultur mit Deponiesickerwasser bei umweltrelevanten Konzentrationen funktioniert und die Zusammensetzungen der gebildeten Organoselenverbindungen derjenigen ahneln, die im Deponiegas gefunden werden, werden diese offensichtlich auch in der Deponie mikrobiologisch gebildet. Empfehlungen und PerspektivenDie mikrobiologische Alkylierung toxischer, anorganischer Selenspezies zu sehr viel weniger problematischen, leichtfluchtigen Organoselenverbindungen stellt eine effiziente Methode dar, um kontaminierte Flachen auf biologischem Wege auch bei relativ niedrigen Konzentrationen zu reinigen.

Some fungal species have been listed as a problem causing fungi in indoor air and most of this group are known to produce mycotoxins. So far, mycotoxins have been found in building materials and in samples representing settled indoor air dust, as well in air samples from industrial or agricultural environments. The present paper presents the results of a mycological study and mycotoxin analyses of dust collected from mechanical ventilation systems in four school buildings in southern Finland. The aim of this work was to answer the question 'Are there mycotoxins in ventilation systems and if so, from where do they originate?' A total of 40 mycotoxins representing indoor and outdoor sources alike were screened in this study, while cultivable fungi were screened using four different cultivation media. Mycotoxins were present in all ventilation systems studied, both in the supply and the exhaust systems examined. The mycotoxins found included satratoxins, verrucarol, trichodermol, enniatins, beauvericin, penicillic acid, sterigmatocystin, chaetoglobosin A, and aflatoxins B1. The mycotoxins were present in minute quantities (pg-ng/g or pg-ng/cm2). The fungal genera associated with respective mycotoxins were found in most of the same sources. Since much the same mycotoxins could be established in both exhaust and supply air systems, it would appear that the mycotoxins found in the schools studied do not for the most part originate from sources within the building but are either normal artefacts of incoming supply air or concentrate or are perhaps produced within the ventilation systems due to infrequent changing of filters and maintenance/cleaning of ventilation ducts and associated parts of the systems.

Selenium adsorbed in the oxyanionic form by Fe-oxides like goethite is considered of benefit for long-term stabilization of (79)Se under near field conditions of radionuclide waste disposal sites. However, microbe-mediated volatilization of the uranium fission product (79)Se has not yet been considered for risk assessment based on the use of the water-solid distribution coefficient K(D). We have performed incubation experiments in a ternary system selenium-microbe-goethite and show that mycobiota including the common black microfungi genera Alternaria alternata are capable of volatilizing the Se even if immobilized by goethite. The microfungi were incubated in a standardized nutrient broth suspension with 10 g L(-1) of the oxide target under defined conditions. Volatile organic selenium (VOSe) species formed in the head space of the culture flasks were sampled and measured directly by a cryotrapping cryofocusing gas chromatographic system coupled with ICP-MS detection (CT-CF-GC-ICP-MS). Alkylated VOSe species were found at the tens to hundreds ng m(-3) levels dominated by dimethyl selenide (DMSe) and dimethyl diselenide (DMDSe). The total amount of DMSe released into the 80-mL headspace volume within the 21 days of incubation was up to 1.12 +/- 0.17 nmol and 0.48 +/- 0.12 nmol for systems without and with goethite amendment, respectively. Alkylation rates of up to 0.1 mumol Se per day and g biomass cannot be neglected as a potential fission product mobilization pathway, unless the inherent radioactivity is proven to prevent any such microbial activity on the long-term. Otherwise it may lead to an onsite accumulation of (79)Se through evapoconcentration in the enclosed underground caverns.

Microbial communities grow more stably when they are associated to surfaces or organized in aggregates. This advantage of biofilms is technically exploited for the degradation of xenobiotics or in biocatalysis, where the fixed biomass has the added advantage of easier separation of excreted products. Whereas the biothermodynamic analysis of growth and product formation of suspended cells is developing fast, there are only few reports on the calorimetric analysis and biothermodynamic interpretation of biofilm evolution. Experiments illustrate the ability of calorimetry to monitor the physiological state of biofilms in real time. Sessile cells of Escherichia coli DH5α DSM 6897 were selected as surrogates of biofilm formers of medical, ecological and biotechnological importance. It is explained how stoichiometric information can be extracted from calorimetric measurements and future possibilities are discussed.

Pheochromocytomas and extra-adrenal paragangliomas (PHEO/PGLs) are rare catecholamine-producing chromaffin cell tumors. For metastatic disease, no effective therapy is available. Overexpression of somatostatin type 2 receptors (SSTR2) in PHEO/PGLs promotes interest in applying therapies using somatostatin analogs linked to radionuclides and/or cytotoxic compounds, such as [(177)Lu]Lu-DOTA-(Tyr(3))octreotate (DOTATATE) and AN-238. Systematic evaluation of such therapies for the treatment of PHEO/PGLs requires sophisticated animal models. In this study, the mouse pheochromocytoma (MPC)-mCherry allograft model showed high tumor densities of murine SSTR2 (mSSTR2) and high tumor uptake of [(64)Cu]Cu-DOTATATE. Using tumor sections, we assessed mSSTR2-specific binding of DOTATATE, AN-238, and somatostatin-14. Therapeutic studies showed substantial reduction of tumor growth and tumor-related renal monoamine excretion in tumor-bearing mice after treatment with [(177)Lu]Lu-DOTATATE compared to AN-238 and doxorubicin. Analyses did not show agonist-dependent receptor downregulation after single mSSTR2-targeting therapies. This study demonstrates that the MPC-mCherry model is a uniquely powerful tool for the preclinical evaluation of SSTR2-targeting theranostic applications in vivo. Our findings highlight the therapeutic potential of somatostatin analogs, especially of [(177)Lu]Lu-DOTATATE, for the treatment of metastatic PHEO/PGLs. Repeated treatment cycles, fractionated combinations of SSTR2-targeting radionuclide and cytotoxic therapies, and other adjuvant compounds addressing additional mechanisms may further enhance therapeutic outcome.

Nondipping blood pressure (BP) is associated with increased morbidity and mortality. This study examines the relationship of "dipping" in 24-hour ambulatory BP monitoring (ABPM) with awake and sleeping urinary norepinephrine (NE) and epinephrine (EPI), and that of urinary NE and EPI with urinary sodium (UNa). Fifty nondippers and 65 dippers were included in the present study. Collected data included age, sex, body mass index, history of hypertension, current antihypertensive treatment, ABPM data, and NE, EPI, and UNa values. Hierarchical multiple regression analysis with the night-to-day ratio (NDR) of systolic BP as a dependent variable showed that the composite term of the NDRs of urinary NE and EPI was a significant predictor for dipping. Results also show a differential role of NE and EPI in circadian UNa excretion in dippers and nondippers. These results indicate that the sympathetic nervous system is involved in the regulation of circadian BP variations and UNa excretion.

Experimental and clinical studies support a protective effect of aldosterone-binding mineralocorticoid receptor (MR) blockade on heart failure. The impact of MR antagonist eplerenone on endothelial function is less well understood. In this study, we analyzed the effects of high-fat diet (HFD) and MR blockade on metabolic parameters and endothelial function in mice overexpressing the endothelial oxidized LDL receptor LOX-1. Mice were fed standard diet (STD) or HFD with or without MR blocker eplerenone for 20 weeks. In agreement with clinical studies aldosterone plasma levels (LC-MS/MS) were elevated in eplerenone-treated mice. Body weight, epididymal, retroperitoneal, mesenteric and perivascular fat were increased after HFD. Diet-induced obesity elevated triglycerides, total cholesterol, and LDL plasma levels. Total cholesterol/HDL cholesterol ratio was increased in HFD fed LOX-1tg mice and decreased by eplerenone. LDLR-/- and HFD-fed LOX1tg and LOX1tg/LDLR-/- (LOLR) mice showed elevated fasting blood glucose levels compared to STD, which were partially normalized by eplerenone. LDLR-/-, LOX 1tg and LOLR mice had impaired endothelial function in thoracic aortas (Mulvany myograph) compared to C57BL/6J mice. HFD pronounced endothelial dysfunction in LOX-1tg and LOLR mice, which was improved by eplerenone. Reactive oxygen species can limit NO availability and were reduced in aorta and adipose tissues after eplerenone. Using a new method to evaluate flow-mediated dilation with a self-developed Optical Coherence Tomography (OCT) device, we could show endothelial dysfunction in saphenous artery in vivo after HFD. This was not restored by eplerenone. In epididymal white adipose tissue of LOX-1tg mice, HFD increased CD206, CD11c, F4/80, leptin, NADPH oxidase (Nox) 1, 2 and 4 and TNF-α mRNA expression (real-time PCR). Eplerenone treatment had no significant impact on CD11c, F4/80 and IL-6 expression, but elevated anti-inflammatory IL-10. CD206, Nox1, Nox2 and Nox4 expression was reduced by eplerenone in mice on HFD. In conclusion, our data support beneficial effects of MR blockade by eplerenone on endothelial function, metabolic parameters, inflammation and oxidative stress in a proatherosclerotic mouse model overexpressing LOX-1 on HFD.

Disruption of the Krebs cycle is a hallmark of cancer. IDH1 and IDH2 mutations are found in many neoplasms, and germline alterations in SDH genes and FH predispose to pheochromocytoma/paraganglioma and other cancers. We describe a paraganglioma family carrying a germline mutation in MDH2, which encodes a Krebs cycle enzyme. Whole-exome sequencing was applied to tumor DNA obtained from a man age 55 years diagnosed with multiple malignant paragangliomas. Data were analyzed with the two-sided Student's t and Mann-Whitney U tests with Bonferroni correction for multiple comparisons. Between six- and 14-fold lower levels of MDH2 expression were observed in MDH2-mutated tumors compared with control patients. Knockdown (KD) of MDH2 in HeLa cells by shRNA triggered the accumulation of both malate (mean ± SD: wild-type [WT] = 1±0.18; KD = 2.24±0.17, P = .043) and fumarate (WT = 1±0.06; KD = 2.6±0.25, P = .033), which was reversed by transient introduction of WT MDH2 cDNA. Segregation of the mutation with disease and absence of MDH2 in mutated tumors revealed MDH2 as a novel pheochromocytoma/paraganglioma susceptibility gene.

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Item does not contain fulltext BACKGROUND: Steroid profiling for diagnosis of endocrine disorders featuring disordered production of steroid hormones is now possible from advances in liquid chromatography with tandem mass spectrometry (LC-MS/MS). Adrenal venous (AV) measurements of aldosterone and cortisol are a standard practice in the clinical work-up of primary aldosteronism, but do not yet take advantage of steroid profiling. METHODS: A novel LC-MS/MS based method was developed for simultaneous measurement of 15 adrenal steroids: aldosterone, corticosterone, 11-deoxycorticosterone, progesterone, pregnenolone, cortisone, cortisol, 11-deoxycortisol, 17-hydroxyprogesterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone-sulfate, 21-deoxycortisol, 18-oxocortisol and 18-hydroxycortisol. These were compared in peripheral venous (pV) and AV plasma from 70 patients undergoing AV sampling with and without cosyntropin stimulation. Aldosterone and cortisol levels measured by LC-MS/MS were compared with those measured by immunoassay. RESULTS: Reproducibility of measurements with coefficients of variation

Pheochromocytoma (PHEO) is a rare but potentially lethal neuroendocrine tumor arising from catecholamine-producing chromaffin cells. Especially for metastatic PHEO, the availability of animal models is essential for developing novel therapies. For evaluating therapeutic outcome in rodent PHEO models, reliable quantification of multiple organ lesions depends on dedicated small-animal in vivo imaging, which is still challenging and only available at specialized research facilities. Here, we investigated whether whole-body fluorescence imaging and monitoring of urinary free monoamines provide suitable parameters for measuring tumor progression in a murine allograft model of PHEO. We generated an mCherry-expressing mouse PHEO cell line by lentiviral gene transfer. These cells were injected subcutaneously into nude mice to perform whole-body fluorescence imaging of tumor development. Urinary free monoamines were measured by liquid chromatography with tandem mass spectrometry. Tumor fluorescence intensity and urinary outputs of monoamines showed tumor growth–dependent increases (P < .001) over the 30 days of monitoring post-tumor engraftment. Concomitantly, systolic blood pressure was increased significantly during tumor growth. Tumor volume correlated significantly (P < .001) and strongly with tumor fluorescence intensity (rs = 0.946), and urinary outputs of dopamine (rs = 0.952), methoxytyramine (rs = 0.947), norepinephrine (rs = 0.756), and normetanephrine (rs = 0.949). Dopamine and methoxytyramine outputs allowed for detection of lesions at diameters below 2.3 mm. Our results demonstrate that mouse pheochromocytoma (MPC)-mCherry cell tumors are functionally similar to human PHEO. Both tumor fluorescence intensity and urinary outputs of free monoamines provide precise parameters of tumor progression in this sc mouse model of PHEO. This animal model will allow for testing new treatment strategies for chromaffin cell tumors.

Background: Comparisons of aldosterone with cortisol during adrenal venous sampling (AVS) provide the currently recommended technique for distinguishing unilateral adenomas from bilateral adrenal hyperplasia during the diagnostic work-up of patients with primary aldosteronism. Selectivity of AVS is routinely established from adrenal venous (AV) to peripheral venous (PV) ratios of cortisol, but is more accurately indicated by AV: PV ratios of plasma metanephrine. Objective: Here we describe a novel liquid-chromatography tandem mass spectrometric (LC-MS/MS) assay for profiling plasma levels of 15 adrenal steroids during AVS, with and without ACTH stimulation. Methods: Selectivity of AVS was established by AV: PV ratios of plasma metanephrine larger than 12. Concentrations of aldosterone, corticosterone, 11-deoxycorticosterone, progesterone, pregnenolone, cortisone, cortisol, 11-deoxycortisol, 17-hydroxy-progesterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone-sulfate, 21-deoxycortisol, 18-oxo-cortisol and 18-hydroxy-cortisol were compared in PV and AV plasma samples from 35 patients with and without ACTH stimulation. Aldosterone and cortisol levels measured by LC-MS/MS were compared with levels measured by immunoassay. Results: Median AV: PV ratios of plasma cortisol (17) were lower than those of corticosterone (52), 11-deoxycorticosterone (68), pregnenolone (72), 11-deoxycortisol (55) and 17-hydroxy-progesterone (57). ACTH stimulation increased AV plasma concentrations of cortisol 6-fold, but had larger effects on other steroids such as corticosterone, which increased by 36-fold. As a result AV plasma concentrations of corticosterone relative to cortisol increased from 6% to 38%. Method comparisons indicated agreement of concentrations of cortisol measured by immunoassay and LC-MS/MS. Conclusion: Compared to other adrenal steroids, cortisol provides a less sensitive marker for selectivity of AVS.

Pheochromocytoma (PHEO) are potentially lethal tumors of adrenal and extra-adrenal chromaffin cells. Available treatment strategies for malignant PHEO are limited. Interestingly, peptide hormone receptors are frequently overexpressed on PHEO and can be used as specific targets for highly effective cytotoxic and radiolabeled anti-tumor peptide analogs. Here, we focused on the establishment and characterization of a novel preclinical fluorescence-based mouse model of PHEO in order to evaluate combined therapies of malignant pheochromocytoma. In previous in vitro studies we demonstrated strong anti-tumor effects of various peptide analogs specifically targeting SSTR2 and LHRH-R in mouse pheochromocytoma cell lines. These neuropeptide receptors were found to be abundantly expressed also on human PHEO samples studied. Furthermore, we examined the expression of these receptors as potential anti-tumor targets in vivo and ex vivo. To this end we established a subcutaneous mouse model of PHEO based on the injection of mCherry-expressing MPCs into the shoulders of athymic nude mice. Tumor development was followed by multimodal in vivo imaging, including fluorescence, PET, CT and MRI. Additionally, we analyzed urinary catecholamines by recently established LC-MS/MS. Both the SSTR2 and LHRH-R are still abundantly expressed in the tumors and could thus be used as therapeutic targets in ongoing pre-clinical studies. In summery, our current work resulted in the establishment of both an appropriate PHEO model system and methodology for our planned combined peptide receptor cytotoxic/radionuclide therapy studies.

Background/objectives Obesity is a major risk factor for the development of type 2 diabetes and other debilitating diseases. Obesity and diabetes are intimately linked with altered levels of adrenal steroids. Elevated levels of these hormones induce insulin resistance and cause cardiovascular diseases. The mechanisms underlying obesity-related alterations in adrenal steroids are still not well understood. Here, we investigated how diet-induced obesity affects the morphology and function of the mouse adrenal cortex. Methods We fed animals either a high-fat diet (HFD) or a normal diet (60% kcal from fat or 10% kcal from fat, respectively) for 18 weeks. We then assessed various aspects of adrenal gland morphology and function, as well as basal plasma concentrations of steroid hormones and ACTH. Results We show that adrenal glands of mice fed a HFD release more corticosterone and aldosterone, resulting in higher plasma levels. This increase is driven by adrenal cortical hyperplasia, and by increased expression of multiple genes involved in steroidogenesis. We demonstrate that diet-induced obesity elevates Sonic hedgehog signaling in Gli1-positive progenitors, which populate the adrenal capsule and give rise to the steroidogenic cells of the adrenal cortex. Feeding animals with a HFD depletes Gli1-positive progenitors, as the adrenal cortex expands. Conclusions This work provides insight into how diet-induced obesity changes the biology of the adrenal gland. The association of these changes with increased Shh signaling suggests possible therapeutic strategies for obesity-related steroid hormone dysfunction.

Absence of the normal 10% reduction of ambulatory blood pressure (ABP) from day to night is associated with morbidity and mortality. The cause of this reduction (or its absence) is not completely understood. We studied excretion of norepinephrine (NE) and epinephrine (EPI) on the same day ABP monitoring was performed in 37 healthy volunteers aged 52±7years, with body mass index (BMI) 25±4 Kg/m 2 , and 24-hour ABP 130±11/82±9 mmHg. Overall SBP decreased from day to night 134±13 to 117±10 mmHg, diastolic blood pressure (DBP) from 86±9 to 72±8 mmHg, and heart rate (HR) from 80±10 to 70±9 beats per minute (bpm). Urinary NE and EPI excretion decreased (p

Background Medication-related interferences with measurements of catecholamines and their metabolites represent important causes of false-positive results during diagnosis of phaeochromocytomas and paragangliomas (PPGLs). Such interferences are less troublesome with measurements by liquid chromatography with tandem mass-spectrometry (LC-MS/MS) than by other methods, but can still present problems for some drugs. Levodopa, the precursor for dopamine used in the treatment of Parkinson’s disease, represents one potentially interfering medication. Methods Plasma and urine samples, obtained from 20 Parkinsonian patients receiving levodopa, were analysed for concentrations of catecholamines and their O-methylated metabolites by LC-MS/MS. Results were compared with those from a group of 120 age-matched subjects and 18 patients with PPGLs. Results Plasma and urinary free and deconjugated (free + conjugated) methoxytyramine, as well as urinary dopamine, showed 22- to 148-fold higher ( P Conclusions These data establish that although levodopa treatment confounds identification of PPGLs that produce dopamine, the therapy is not a problem for use of LC-MS/MS measurements of plasma and urinary normetanephrine and metanephrine to diagnose more commonly encountered PPGLs that produce noradrenaline or adrenaline.

Objective: To determine whether the accumulation of 2-hydroxyglutarate in IDH-mutated low-grade gliomas WHO grade II (LGG) correlates with their malignant transformation and to evaluate changes in metabolite levels during malignant progression. Method: Samples from 54 patients were screened for IDH[for full text, please go to the a.m. URL], 64. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC)

Adrenal vein sampling is used to establish the origins of excess production of adrenal hormones in primary aldosteronism. Correct catheter positioning is confirmed using adrenal vein measurements of cortisol, but this parameter is not always reliable. Plasma metanephrine represents an alternative parameter. The objective of our study was to determine the use of plasma metanephrine concentrations to establish correct catheter positioning during adrenal vein sampling with and without cosyntropin stimulation. We included 52 cosyntropin-stimulated and 34 nonstimulated sequential procedures. Plasma cortisol and metanephrine concentrations were measured in adrenal and peripheral venous samples. Success rates of sampling, using an adrenal to peripheral cortisol selectivity index of 3.0, were compared with success rates of metanephrine using a selectivity index determined by receiver operating characteristic curve analysis. Among procedures assessed as selective using cortisol, the adrenal to peripheral vein ratio of metanephrine was 6-fold higher than that of cortisol (94.0 versus 15.5; P P

Background Measurements of plasma normetanephrine (NMN) and metanephrine (MN) provide a sensitive test for diagnosis of phaeochromocytomas and paragangliomas (PPGLs), but do not allow detection of dopamine-producing tumours. Here we introduce a novel mass spectrometric based method coupled to ultraperformance liquid chromatography (LC-MS/MS) for measuring NMN, MN and 3-methoxytyramine (MTY), the O-methylated metabolite of dopamine. Methods Specific collision-induced fragment ions assessed by multireaction monitoring transitions were used for identification, with quantification according to signal intensities of analytes relative to stable isotope labelled internal standards. Results for solid-phase extracted samples from 196 subjects analysed by LC-MS/MS were compared with those analysed by liquid chromatography with electrochemical detection (LC-ECD). Concentration ranges in 125 volunteers were compared with those from 63 patients with PPGLs, including 14 with metastatic disease. Results The LC-MS/MS method showed linearity over four orders of magnitude with analytical sensitivity sufficient to measure to 0.02 nmol/L. Intra- and inter-assay coefficients of variation ranged from 2.8% to 13.5%. NMN and MN were respectively measured 17% and 10% higher and MTY 26% lower by LC-MS/MS than by LC-ECD. Medians and ranges for 3-methoxytramine, NMN and MN were respectively 0.08 (0.03-0.13), 0.35 (0.13-0.95) and 0.15 (0.07-0.33) nmol/L in volunteers. Among patients with PPGLs, plasma methoxytyramine was six-fold higher in patients with than without metastastases (1.09 versus 0.19 nmol/L) and in three patients was the only metabolite increased. Conclusions The LC-MS/MS method enables accurate, selective and sensitive measurements of catecholamine O-methylated metabolites that should be particularly useful for screening and management of dopamine-producing metastatic PPGLs.

International audience; CONTEXT: Recommendations have not been established concerning imaging to screen SDHx mutation carriers for paraganglioma and pheochromocytoma.OBJECTIVE: Our objective was to compare the performance of gadolinium-enhanced magnetic resonance angiography, contrast-enhanced computed tomography, and [(123)I]metaiodo-benzylguanidine and somatostatin receptor scintigraphies for detecting head and neck and thoracic-abdominal-pelvic paragangliomas in SDHx mutation carriers.DESIGN AND SETTING: We conducted a prospective, multicenter study from June 2005 to December 2009 at 23 French medical centers.PATIENTS: A total of 238 index cases or relatives carrying mutations in SDHD, SDHB, or SDHC genes were included.INTERVENTION: Images obtained by each technique were analyzed blind, without knowledge of results from other tests, first in each local center and then centrally.MAIN OUTCOME MEASURES: We evaluated sensitivity, specificity, and likelihood ratios for individual and combinations of tests, the gold standard being the consensus of an expert committee.RESULTS: Two hundred two tumors were diagnosed in 96 subjects. At local assessment, the sensitivity of anatomical imaging for detecting all tumors was higher (85.7%) than that of both scintigraphic techniques (42.7% for [(123)I]metaiodo-benzylguanidine and 69.5% for somatostatin receptor scintigraphy), except for thoracic localizations where somatostatin receptor scintigraphy was more sensitive (61.5 vs. 46.2% for anatomical imaging and 30.8% for [(123)I]metaiodo-benzylguanidine scintigraphy). The best diagnostic performance during local assessment was obtained by combining anatomical imaging tests and somatostatin receptor scintigraphy (sensitivity 91.7%). Central assessment significantly increased the sensitivity (98.6%) of tests in combination.CONCLUSIONS: In routine practice, the imaging work-up for screening SDHx mutation carriers should include thoraco-abdomino-pelvic computed tomography, head and neck magnetic angiography, and somatostatin receptor scintigraphy. Expert centralized image assessment is recommended.

IDH1 R132H mutations are considered to play a key role in the development of low grade gliomas and therefrom derived secondary glioblastomas (GBM). Wild type IDH1 converts isocitrate to α-ketoglutarate (a-KG) while reducing NADP+. IDH1R132H has a neomorphic enzymatic function using a-KG to generate high amounts of the oncometabolite D-2-hydroxyglutarate (D-2-HG). While the effects of D-2-HG have been subject to intense research, D-2-HG independent effects of IDH1R132H on energy homeostasis and redox status are not well studied. Here we demonstrate that IDH1R132H transduction but not D-2-HG alone leads to significantly decreased Krebs cycle metabolite concentrations and proliferation in U87 and the primary GBM cell line HT7606 as well as in immortalized astrocytes SVGp12. Furthermore, IDH1R132H mutation, but not D-2-HG treatment, resulted in a significant drop in NADPH levels in tumor cells (U87 and HT7606), whereas immortalized astrocytes retained normal NADPH levels. Since NAPDH levels can be restored via the reaction of the NAD-kinase, we analyzed NAD levels and enzymes involved in NAD-synthesis in our cell lines. Indeed we found a significant drop of NAD levels and in the activity of the NAD-dependent enzyme sirtuin in IDH1R132H mutant U87 and HT7606 but not in NADPH stable SVGp12-IDH1R132H. Interestingly, there were marked differences in expression of NAD-synthesis enzymes between the different cell-lines. In particular, NAMPT-levels were much higher in U87 and HT7606 then in astrocytes (SVGp12) and significantly decreased in U87-IDH1R132H and HT7606-IDH1R132H. Importantly, we also found decreased levels of NAMPT in primary tumor tissues and patient derived glioma cell lines with IDH1 R132H compared to wild type gliomas. Altogether our results for the first time show that the IDH1 mutation directly affects energy homeostasis and redox status in a cell-type dependent manner. We hypothesize that this leads to a drop in NADPH and NAD-levels during malignant progression, resulting in a disadvantage for proliferating tumor cells. This is in line with the favorable prognosis and good response to chemo- and radiation therapy clinically observed in IDH-mutated gliomas. Our findings suggest that the impaired metabolism in IDH1-mutant tumors might be a promising target for future therapies. Citation Format: Evelin Schrock, Julia Biedermann, Khalil Abou-El-Ardat, Matthias Lehmann, Marina Conde, Mirko Peitzsch, Susan Richter, Ralf Wiedemuth, Matthias Meinhardt, William P. J. Leenders, Christel Herold-Mende, Graeme Eisenhofer, Simone P. Niclou, Leoni Kunz-Schughart, Achim Temme, Barbara Klink. Effects of the IDH1 R132H mutation on redox status and metabolism are cell type dependent but independent from D-2-hydroxyglutarate accumulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-308.

Objective We introduce a novel liquid chromatographic tandem-mass spectrometric method for simultaneous measurements of urinary catecholamines and their free O-methylated metabolites, which we compare to the deconjugated metabolites. Methods Method performance was validated for recovery, linearity, precision and accuracy, analyte stability, ion suppression and carry over. Results from 53 patients with and 138 volunteers without pheochromocytoma were compared. Results Analyte recoveries ranged from 60 to 96% and intra- and inter-assay coefficients of variation from 2.7 to 13.2%. The method showed excellent linearity over 3 orders of magnitude with analytical sensitivity sufficient to measure to 1.2 nmol/L. Free O-methylated metabolites were excreted at less than 20% the rates of the deconjugated metabolites, but were easily measureable. Increases in urinary normetanephrine in pheochromocytoma patients relative to volunteers were higher for free than deconjugated metabolites and higher for both than for norepinephrine (10 vs 5.5 vs 3.7 fold increases). In contrast, relative increases in urinary free versus deconjugated metanephrine (2.7 and 3.2) and methoxytyramine (2.1 and 1.9) did not differ, but for methoxytyramine were larger than for dopamine (1.2). Conclusion Measurements of urinary catecholamines and their free O-methylated metabolites by our method provide potential advantages over urinary deconjugated metanephrines for diagnosis of pheochromocytoma.

Here, we describe a novel method utilizing double stable isotope ultra performance liquid chromatography-tandem mass spectrometry to measure tissue contents and activity of phenylethanolamine N-methyltransferase (PNMT), the enzyme responsible for synthesis of the stress hormone, epinephrine. The method is based on measurement of deuterium-labeled epinephrine produced from the reaction of norepinephrine with deuterium-labeled S-adenosyl-l-methionine as the methyl donor. In addition to enzyme activity, the method allows for determination of tissue contents of PNMT using human recombinant enzyme for calibration. The calibration curve for epinephrine was linear over the range of 0.1 to 5,000 pM, with 0.5 pM epinephrine representing the lower limit of quantification. The calibration curve relating PNMT to production of deuterium-labeled epinephrine was also linear from 0.01 to 100 ng PNMT. Intra- and inter-assay coefficients of variation were respectively 12.8 % (n = 10) and 10.9 to 13.6 % (n = 10). We established utility of the method by showing induction of the enzyme by dexamethasone in mouse pheochromocytoma cells and strong relationships to PNMT gene expression and tissue epinephrine levels in human pheochromocytomas. Development of this assay provides new possibilities for investigations focusing on regulation of PNMT, the crucial final enzyme responsible for synthesis of epinephrine, the primary fight-or-flight stress hormone.

We compared the efficiency of some commercially available products and methods used for remediation of mould-contaminated building materials. Samples of gypsum board and pinewood were artificially contaminated with toxin-producing isolates of Stachybotrys chartarum and Aspergillus versicolor, respectively, then, ten different remediation treatments were applied according to the manufacturers' instructions. Microbial and chemical analyses of the infested materials were carried out both immediately before and after treatment, after six weeks of drying at room temperature, and after another six weeks of remoistening. The aim of the study was to determine whether the investigated methods could inhibit the mould growth and destroy some selected mycotoxins produced by the moulds. None of the decontamination methods tested could completely eliminate viable moulds. Some methods, especially boron based chemicals, ammonium based chemicals, and oxidation reduced the contents of mycotoxins produced by S. chartarum (satratoxin G and H, verrucarol), whereas the one which uses an ammonium based chemical reduced the amount of sterigmatocystin produced by A. versicolor with statistical significance. No remediation treatment eliminated all the toxins from the damaged materials. These results emphasize the importance to work preventively with moisture safety throughout the construction processes and management to prevent mould growth on building materials.

INTRODUCTION: Diffuse gliomas are the most prevalent brain tumors in adults and cannot be cured. Amino-acid substitution at position 132 of cytoplasmic enzyme isocitrate dehydrogenase 1 (IDH1) are characteristic for lower grade gliomas and therefrom derived secondary glioblastomas (GBM). The most frequent mutation observed in low grade gliomas is IDH1 c.395G>A (p.R132H). The wild type IDH1 converts isocitrate to α-ketoglutarate while reducing nicotinamide adenine dinucleotide phosphate (NADP). In contrast, IDH1 R132H produces high amounts of 2-hydroxyglutarate (2HG), which leads to a hypermethylation phenotype and is therefore considered to be an oncometabolite. Nevertheless, the accumulation of 2HG cannot explain all the observed phenomena in gliomas. We aimed to investigate other mechanisms by which IDH1 R132H mutation contributes to gliomagenesis. MATERIALS AND METHODS: We have stably transduced three cell lines (a established GBM cell line, a primary patient-derived GBM cell line, and an immortalized astrocyte cell line) with IDH1 c.395G>A cDNA using viral vectors. Transduction success was validated on DNA, RNA and protein levels using Sanger sequencing, allele-specific PCR and Western blots. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to measure Krebs cycle metabolite concentrations. In order to find effects independent of 2HG, we added the oncometabolite to empty vector controls. NADP+/NADPH quantification kits were used to determine the concentrations of this coenzyme. RESULTS: We confirmed the presence of IDH1 c.395G>A;p.R132H mutation in the cells transduced with the mutant variant of IDH1. There was an up to 1700-fold increase of the oncometabolite 2HG in the cells with the mutation compared to an empty vector control. The levels of glutamate significantly decreased in cells with the IDH1 mutation as well as in empty vector controls after 2HG treatment. We found a ∼50% decrease in NADPH concentration in GBM cells with the IDH1 mutation but not in astrocytes. Interestingly, there was a decrease in proliferation of GBM cells with IDH1 R132H. This effect was confirmed in 3D spheroid cell cultures. CONCLUSION: The mechanisms of how IDH1 R132H mutations contribute to gliomagenesis are still far from clear. We found that mutations in IDH1 lead to changes in Krebs cycle metabolite concentrations and energy homeostasis while decreasing proliferation in vitro. We are currently analyzing the effect of IDH1 R132H on the transcriptome level in comparison with IDH1 wt cells treated with 2HG. Based on these profiles we aim to investigate possible genetic mechanisms which are independet of the oncometabolite 2HG. These results will also be presented.

Context Catecholamines and adrenocortical steroids are important regulators of blood pressure. Bidirectional relationships between adrenal steroids and catecholamines have been established but whether this is relevant to patients with pheochromocytoma is unclear. Objective This study addresses the hypothesis that patients with pheochromocytoma and paraganglioma (PPGL) have altered steroid production compared with patients with primary hypertension. Design Multicenter cross-sectional study. Setting Twelve European referral centers. Patients Subjects included 182 patients with pheochromocytoma, 36 with paraganglioma and 270 patients with primary hypertension. Patients with primary aldosteronism (n = 461) and Cushing syndrome (n = 124) were included for additional comparisons. Intervention In patients with PPGLs, surgical resection of tumors. Outcome measures Differences in mass spectrometry–based profiles of 15 adrenal steroids between groups and after surgical resection of PPGLs. Relationships of steroids to plasma and urinary metanephrines and urinary catecholamines. Results Patients with pheochromocytoma had higher (P < .05) circulating concentrations of cortisol, 11-deoxycortisol, 11-deoxycorticosterone, and corticosterone than patients with primary hypertension. Concentrations of cortisol, 11-deoxycortisol, and corticosterone were also higher (P < .05) in patients with pheochromocytoma than with paraganglioma. These steroids correlated positively with plasma and urinary metanephrines and catecholamines in patients with pheochromocytoma, but not paraganglioma. After adrenalectomy, there were significant decreases in cortisol, 11-deoxycortisol, corticosterone, 11-deoxycorticosterone, aldosterone, and 18-oxocortisol. Conclusions This is the first large study in patients with PPGLs that supports in a clinical setting the concept of adrenal cortical–medullary interactions involving an influence of catecholamines on adrenal steroids. These findings could have implications for the cardiovascular complications of PPGLs and the clinical management of patients with the tumors.

Unilateral primary aldosteronism (PA) is the most common surgically curable form of hypertension that must be accurately differentiated from bilateral PA for therapeutic management (surgical versus medical). Adrenalectomy results in biochemical cure (complete biochemical success) in almost all patients diagnosed with unilateral PA; the remaining patients with partial or absent biochemical success comprise those with persisting aldosteronism who were misdiagnosed as unilateral PA preoperatively. To identify determinants of postsurgical biochemical outcomes, we compared the adrenal histopathology and the peripheral venous steroid profiles of patients with partial and absent or complete biochemical success after adrenalectomy for unilateral PA. A large multicenter cohort of adrenals from patients with absent and partial biochemical success (n=43) displayed a higher prevalence of hyperplasia (49% versus 21%; P =0.004) and a lower prevalence of solitary functional adenoma (44% versus 79%; P

Contains fulltext : 137037.pdf (Publisher’s version ) (Closed access) OBJECTIVE: To document the influences of blood sampling under supine fasting versus seated nonfasting conditions on diagnosis of phaeochromocytomas and paragangliomas (PPGL) using plasma concentrations of normetanephrine, metanephrine and methoxytyramine. DESIGN AND METHODS: Biochemical testing for PPGL was performed on 762 patients at six centres, two of which complied with requirements for supine sampling after an overnight fast and four of which did not. Phaeochromocytomas and paragangliomas were found in 129 patients (67 noncompliant, 62 compliant) and not in 633 patients (195 noncompliant, 438 compliant). RESULTS: Plasma concentrations of normetanephrine and methoxytyramine did not differ between compliant and noncompliant sampling conditions in patients with PPGL but were 49-51% higher in patients without PPGL sampled under noncompliant compared with compliant conditions. The 97.5 percentiles of distributions were also higher under noncompliant compared with compliant conditions for normetanephrine (1.29 vs 0.79 nmol/l), metanephrine (0.49 vs 0.41 nmol/l) and methoxytyramine (0.42 vs 0.18 nmol/l). Use of upper cut-offs established from seated nonfasting sampling conditions resulted in substantially decreased diagnostic sensitivity (98% vs 85%). In contrast, use of upper cut-offs established from supine fasting conditions resulted in decreased diagnostic specificity for testing under noncompliant compared with compliant conditions (71% vs 95%). CONCLUSIONS: High diagnostic sensitivity of plasma normetanephrine, metanephrine and methoxytyramine for the detection of PPGL can only be guaranteed using upper cut-offs of reference intervals established with blood sampling under supine fasting conditions. With such cut-offs, sampling under seated nonfasting conditions can lead to a 5.7-fold increase in false-positive results necessitating repeat sampling under supine fasting conditions.

Novel particle therapy was implemented into standard-of-care for cancer patients during the last years. However, experimental studies investigating cellular and molecular mechanisms are lacking and prognostic biomarker are urgently needed. Cancer stem cell (CSC)-related biomarkers such as aldehyde dehydrogenase (ALDH) are known to cellular radiosensitivity by affecting defense against reactive oxygen species, DNA damage repair and cell survival. Within a previous study, we found that ionizing radiation itself enriches for ALDH-positive CSCs. Within the present study, we investigated CSC marker dynamics in prostate cancer, head and neck cancer and glioblastoma cells upon proton beam irradiation. We found that proton irradiation has an increased CSC targeting potential, reduced methylation of activating histone marks and a lower induction of cellular senescence compared to conventional photon irradiation. Interestingly, mathematical modeling indicated to differences in plasticity rates among ALDH-positive CSCs and ALDH-negative cancer cells between the two irradiation types.