Metabolic Adaptation as Potential Target in Papillary Renal Cell Carcinomas Based on Their In Situ Metabolic Characteristics.

Metabolic characteristics of kidney cancers have mainly been obtained from the most frequent clear cell renal cell carcinoma (CCRCC) studies. Moreover, the bioenergetic perturbances that affect metabolic adaptation possibilities of papillary renal cell carcinoma (PRCC) have not yet been detailed. Therefore, our study aimed to analyze the in situ metabolic features of PRCC vs. CCRCC tissues and compared the metabolic characteristics of PRCC, CCRCC, and normal tubular epithelial cell lines. The protein and mRNA expressions of the molecular elements in mammalian target of rapamycin (mTOR) and additional metabolic pathways were analyzed in human PRCC cases compared to CCRCC. The metabolic protein expression pattern, metabolite content, mTOR, and metabolic inhibitor sensitivity of renal carcinoma cell lines were also studied and compared with tubular epithelial cells, as "normal" control. We observed higher protein expressions of the "alternative bioenergetic pathway" elements, in correlation with the possible higher glutamine and acetate consumption in PRCC cells instead of higher glycolytic and mTOR activity in CCRCCs. Increased expression of certain metabolic pathway markers correlates with the detected differences in metabolite ratios, as well. The lower lactate/pyruvate, lactate/malate, and higher pyruvate/citrate intracellular metabolite ratios in PRCC compared to CCRCC cell lines suggest that ACHN (PRCC) have lower Warburg glycolytic capacity, less pronounced pyruvate to lactate producing activity and shifted OXPHOS phenotype. However, both studied renal carcinoma cell lines showed higher mTOR activity than tubular epithelial cells cultured in vitro, the metabolite ratio, the enzyme expression profiles, and the higher mitochondrial content also suggest increased importance of mitochondrial functions, including mitochondrial OXPHOS in PRCCs. Additionally, PRCC cells showed significant mTOR inhibitor sensitivity and the used metabolic inhibitors increased the effect of rapamycin in combined treatments. Our study revealed in situ metabolic differences in mTOR and metabolic protein expression patterns of human PRCC and CCRCC tissues as well as in cell lines. These underline the importance in the development of specific new treatment strategies, new mTOR inhibitors, and other anti-metabolic drug combinations in PRCC therapy.

Wnt-Signaling Regulated by Glucocorticoid-Induced miRNAs.

Glucocorticoids (GCs) are pleiotropic hormones which regulate innumerable physiological processes. Their comprehensive effects are due to the diversity of signaling mechanism networks. MiRNAs, small, non-coding RNAs contribute to the fine tuning of signaling pathways and reciprocal regulation between GCs and miRNAs has been suggested. Our aim was to investigate the expressional change and potential function of GC mediated miRNAs. The miRNA expression profile was measured in three models: human adrenocortical adenoma vs. normal tissue, steroid-producing H295R cells and in hormonally inactive HeLa cells before and after dexamethasone treatment. The gene expression profile in 82 control and 57 GC-affected samples was evaluated in GC producing and six different GC target tissue types. Tissue-specific target prediction (TSTP) was applied to identify the most relevant miRNA-mRNA interactions. Glucocorticoid treatment resulted in cell type-dependent miRNA expression changes. However, 19.5% of the influenced signaling pathways were common in all three experiments, of which the Wnt-signaling pathway seemed to be the most affected. Transcriptome data and TSTP showed similar results, as the Wnt pathway was significantly altered in both the GC-producing adrenal gland and all investigated GC target tissue types. In different cell types, different miRNAs led to the regulation of similar pathways. Wnt signaling may be one of the most important signaling pathways affected by hypercortisolism. It is, at least in part, regulated by miRNAs that mediate the glucocorticoid effect. Our findings on GC producing and GC target tissues suggest that the alteration of Wnt signaling (together with other pathways) may be responsible for the leading symptoms observed in Cushing's syndrome.

Gamma-Synuclein Levels Are Elevated in Peritoneal Fluid of Patients with Endometriosis.

BACKGROUND The role of gamma-synuclein (SNCG) has been widely examined in malignant conditions due to its possible role in disease progression, but very little information is available on its theoretical function on endometriosis formation. MATERIAL AND METHODS Between January 2016 and December 2016, we collected peritoneal fluid and plasma samples from 45 consecutive female patients, of which 15 were without endometriosis, 15 had minimal to mild endometriosis, and 15 had moderate to severe endometriosis. The statistical power was 0.98. We evaluated SNCG levels in the peritoneal fluid and plasma of patients diagnosed with endometriosis, and we compared them with the levels obtained from disease-free control subjects by using enzyme-linked immunosorbent assay. RESULTS SNCG levels were statistically significantly (1.2-fold) higher in the peritoneal fluid of patients with endometriosis compared to controls (p=0.04). We did not find a significant difference between SNCG levels in the plasma of our endometriosis patients and the control group (p=0.086). However, despite previous data showing very limited expression of SNCG in healthy tissues, we found SNCG in the peritoneal fluid of all of the patients in our healthy control group. CONCLUSIONS Levels of SNCG were statistically significantly higher in the peritoneal fluid of patients with endometriosis compared to disease-free controls, which may indicate its possible role the formation and progression of the disease. Moreover, its biological function should be further investigated due to the conflicting results concerning its expression in healthy tissues.

Glucocorticoids Influencing Wnt/ß-Catenin Pathway; Multiple Sites, Heterogeneous Effects.

Glucocorticoid hormones are vital; their accurate operation is a necessity at all ages and in all life situations. Glucocorticoids regulate diverse physiological processes and they use many signaling pathways to fulfill their effect. As the operation of these hormones affects many organs, the excess of glucocorticoids is actually detrimental to the whole human body. The endogenous glucocorticoid excess is a relatively rare condition, but a significant proportion of adult people uses glucocorticoid medication for the treatment of chronic illnesses, therefore they are exposed to the side effects of long-term glucocorticoid treatment. Our review summarizes the adverse effects of glucocorticoid excess affecting bones, adipose tissue, brain and skin, focusing on those effects which involve the Wnt/ß-catenin pathway.

Targeting cellular metabolism using rapamycin and/or doxycycline enhances anti-tumour effects in human glioma cells.

BACKGROUND: Glioma is the most common highly aggressive, primary adult brain tumour. Clinical data show that therapeutic approaches cannot reach the expectations in patients, thus gliomas are mainly incurable diseases. Tumour cells can adapt rapidly to alterations during therapeutic treatments related to their metabolic rewiring and profound heterogeneity in tissue environment. Renewed interests aim to develop effective treatments targeting angiogenesis, kinase activity and/or cellular metabolism. mTOR (mammalian target of rapamycin), whose hyper-activation is characteristic for many tumours, promotes metabolic alterations, macromolecule biosynthesis, cellular growth and survival. Unfortunately, mTOR inhibitors with their lower toxicity have not resulted in appreciable survival benefit. Analysing mTOR inhibitor sensitivity, other metabolism targeting treatments and their combinations could help to find potential agents and biomarkers for therapeutic development in glioma patients. METHODS: In vitro proliferation assays, protein expression and metabolite concentration analyses were used to study the effects of mTOR inhibitors, other metabolic treatments and their combinations in glioma cell lines. Furthermore, mTOR activity and cellular metabolism related protein expression patterns were also investigated by immunohistochemistry in human biopsies. Temozolomide and/or rapamycin treatments altered the expressions of enzymes related to lipid synthesis, glycolysis and mitochondrial functions as consequences of metabolic adaptation; therefore, other anti-metabolic drugs (chloroquine, etomoxir, doxycycline) were combined in vitro. RESULTS: Our results suggest that co-targeting metabolic pathways had tumour cell dependent additive/synergistic effects related to mTOR and metabolic protein expression patterns cell line dependently. Drug combinations, especially rapamycin + doxycycline may have promising anti-tumour effect in gliomas. Additionally, our immunohistochemistry results suggest that metabolic and mTOR activity alterations are not related to the recent glioma classification, and these protein expression profiles show individual differences in patients' materials. CONCLUSIONS: Based on these, combinations of different new/old drugs targeting cellular metabolism could be promising to inhibit high adaptation capacity of tumour cells depending on their metabolic shifts. Relating to this, such a development of current therapy needs to find special biomarkers to characterise metabolic heterogeneity of gliomas.

[Extraadrenal glucocorticoid synthesis]. / Extraadrenalis glükokortikoidszintézis.

Endogenous glucocorticoids exert a diverse array of physiological processes and play an important role in immune modulatory and anti-inflammatory responses. The secretion of cortisol by the adrenal gland is regulated through two mechanisms. Systemic regulation is substantiating by the hypothalamo-pituitary-adrenal axis. Furthermore, a tissue-specific local regulatory system, containing the 11ß-hydroxysteroid dehydrogenase enzyme responsible for local glucocorticoid synthesis and the glucocorticoid receptor, has also been demonstrated. Based on the recent evidences, an extra-adrenal corticosteroid synthesis exists in various tissues. Steroidogenic enzymes necessary for this de novo corticosteroid synthesis have been observed in the skin, intestine, thymus and possibly in the brain, heart and lung. These locally synthesized steroids most likely act in an autocrine and paracrine manner and their regulation is mediated by local regulatory loops. The importance of this de novo corticosteroid synthesis seems to be important in the regulation of local homeostasis, immune processes and tissue-specific inflammatory reactions. Orv Hetil. 2018; 159(7): 260-268.

[Steroid 21-hydroxylase deficiency, the most frequent cause of congenital adrenal hyperplasia]. / Szteroid-21-hidroxiláz-deficientia, a congenitalis adrenalis hyperplasia leggyakoribb oka.

Congenital adrenal hyperplasia is a group of genetic diseases due to the disablement of 7 genes; one of them is steroid 21-hydroxylase deficiency. The genes of congenital adrenal hyperplasia encode enzymes taking part in the steroidogenesis of adrenal gland. Steroid 21-hydroxylase deficiency is an autosomal recessive disorder caused by mutations of the steroid 21-hydroxylase gene. The mutations of steroid 21-hydroxylase gene cause 95% of the congenital adrenal hyperplasia cases. Although the non-classic steroid 21-hydroxylase deficiency with mild symptoms is seldom diagnosed, the classic steroid 21-hydroxylase deficiency may lead to life-threatening salt-wasting and adrenal crises due to the insufficient aldosterone and cortisol serum levels. The classic type requires life-long steroid replacement which may result in cushingoid side effects, and typical comorbidities may be also developed. The patients' quality of life is decreased, and their mortality is much higher than that of the population without steroid 21-hydroxylase deficiency. The diagnosis, consequences and the patients' life-long clinical care require a multidisciplinary approach: the specialists in pediatrics, internal medicine, endocrinology, laboratory medicine, genetic diagnostics, surgery, obstetrics-gynecology and psychology need to work together. Orv Hetil. 2018; 159(7): 269-277.

Looking beyond linear regression and Bland-Altman plots: a comparison of the clinical performance of 25-hydroxyvitamin D tests.

BACKGROUND: The systematic evaluation of the clinical concordance of various 25-hydroxyvitamin D (25OHD) testing methods is presented. The need for this approach is raised by the discrepancies in the analytical performance of the available assays. METHODS: The analytical and clinical performance of six automated 25OHD assays and an in-house liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was investigated. Leftover serum samples (n=162, SA: n=114) were analyzed and all 21 assay combinations were evaluated. The utility of Cohen's κ values was assessed by transforming them into minimum percentage agreement (MPA). McNemar's hypothesis test was employed for testing the symmetry of the disagreeing classification outcomes within each method pair. RESULTS: Depending on the assay method, the ratio of results classified as positive (<20 ng/mL) was 13.5%-40.0%. The percentage agreement (PA) was 74.1%-92.6%. Compared to other methods, significantly more hypovitaminosis cases were delivered by DiaSorin Liaison® 25 OH vitamin D Total (DL) and significantly fewer by IDS-iSYS 25-Hydroxy Vitamin DS (II). The strongest clinical concordance was exerted by II vs. LC-MS/MS. The κ-derived MPA showed close similarity to the PA scores. McNemar's tests confirmed the asymmetry of the disagreement in the classification in 14 method combinations. CONCLUSIONS: The presented approach allows the prediction of the clinical consequences of a 25OHD method transfer. Differences in the clinical classification of assay results are likely encountered when transferring to a new method, even between assays standardized according to the Vitamin D Standardization Program (VDSP) Reference Method Procedure (RMP).

[The biological and clinical relevance of estrogen metabolome]. / Az ösztrogénmetabolom biológiai és klinikai jelentosége lokális folyamatokban.

Considerable knowledge has been gathered on the physiological role of estrogens. However, fairly little information is available on the role of compounds produced in the breakdown process of estrone and estradiol wich may play a role in various diseases associated with estrogen impact. To date, approximately 15 extragonadal estrogen-related compounds have been identified. These metabolites may exert protective, or, instead, pro-inflammatory and/or pro-oncogenic activity in a tissue-specific manner. Systemic and local estrogen metabolite levels are not necesserily correlated, which may promote the diagnostic significance of the locally produced estrogen metabolites in the future. The aim of the present study is a bibliographic review of the extragonadal metabolome in peripheral tissues, and to highlight the role of the peripheral tissue homeostasis of estrogens as well as the non-hormonal biological activity and clinical significance of the estrogen metabolome. Orv Hetil. 2017; 158(24): 929-937.

[Focusing on tissue biomarkers. Estrogens as key players in the immune response and autoimmunity]. / Fókuszban a szöveti biomarkerek. Az ösztrogének mint a szövetspecifikus immunválasz és autoimmunitás modulálásának kulcsszereploi.

Estrogens modulate the immune response as well as the risk and progression of autoimmune disorders. Their effects are mediated by nuclear receptors (i.e. estrogen receptor alpha and beta), membrane receptors, and are influenced by their interactions with other hormones. Locally produced hormones and cytokines are the main factors in maintaining tissue homeostasis. The response of immune cells to estrogens is related to their developmental stage. The diverse effects of estrogens on various autoimmune disorders are the result of the versatility of their pathomechanism. In general, progression of B-cell mediated disorders is aggravated by estrogens. Their effects on T-cell mediated disorders, on the other hand, are driven by Th1 or Th2 dominance. As estrogens promote the escalation of the Th2 immune response, Th2-dominant disorders are aggravated, while Th1-dominant disorders are ameliorated upon high estrogen levels. Inflammation on its own also modulates the impact of estrogens. Inflammatory cytokines alter the expression of the alpha and beta estrogen receptors as well as the activity of estrogen metabolizing enzymes. Monitoring the local, tissue-wide interaction between hormones and immune cells would provide a better tool for identification and characterization of molecules involved in this system. To date, routinely used laboratory methods have a limited role in monitoring the local effects of estrogens. In this current paper the authors summarize the role of estrogens in immune system and overview those novel methods which are useful in the investigation of local endocrine milieu.

Aspirin Mediates Its Antitumoral Effect Through Inhibiting PTTG1 in Pituitary Adenoma.

CONTEXT: DNA demethylation and inhibitory effects of aspirin on pituitary cell proliferation have been demonstrated. OBJECTIVE: Our aim was to clarify the molecular mechanisms behind the aspirin-related effects in pituitary cells. METHODS: DNA methylome and whole transcriptome profile were investigated in RC-4B/C and GH3 pituitary cell lines upon aspirin treatment. Effects of aspirin and a demethylation agent, decitabine, were further tested in vitro. PTTG1 expression in 41 human PitNET samples and whole genome gene and protein expression data of 76 PitNET and 34 control samples (available in Gene Expression Omnibus) were evaluated. RESULTS: Aspirin induced global DNA demethylation and consequential transcriptome changes. Overexpression of Tet enzymes and their cofactor Uhrf2 were identified behind the increase of 5-hydroxymethylcytosine (5hmC). Besides cell cycle, proliferation, and migration effects that were validated by functional experiments, aspirin increased Tp53 activity through p53 acetylation and decreased E2f1 activity. Among the p53 controlled genes, Pttg1 and its interacting partners were downregulated upon aspirin treatment by inhibiting Pttg1 promoter activity. 5hmC positively correlated with Tet1-3 and Tp53 expression, and negatively correlated with Pttg1 expression, which was reinforced by the effect of decitabine. Additionally, high overlap (20.15%) was found between aspirin-regulated genes and dysregulated genes in PitNET tissue samples. CONCLUSION: A novel regulatory network has been revealed, in which aspirin regulated global demethylation, Tp53 activity, and Pttg1 expression along with decreased cell proliferation and migration. 5hmC, a novel tissue biomarker in PitNET, indicated aspirin antitumoral effect in vitro as well. Our findings suggest the potential beneficial effect of aspirin in PitNET.

A relative quantitation method for measuring DNA methylation and hydroxymethylation using guanine as an internal standard.

Global DNA methylation and hydroxymethylation play an important role in gene expression. They can be connected with several diseases. The modification status could be a biomarker to determine the status of disease. A fast, easy and accurate liquid chromatography - tandem mass spectrometry method has been developed for the precise quantitation of 5-methylcytosine and 5-hydroxymethylcytosine. Formic acid was used for the hydrolysis of the DNA strand resulting in nucleobases. These polar hydrolysis products were separated on a normal phase column using reversed phase eluents in inverse gradient mode. Multiple reaction monitoring was applied to achieve high selectivity and sensitivity for the quantitation. A new relative quantitation model was developed by using guanine, as an internal standard, present in samples. The new method was successfully validated with excellent accuracy and precision values in the range of 0.005-0.5% for 5hmC and 1-15% for 5mC. The main advantages of this quantitation method are that, due to relative quantitation, calibration curves can be used without reacquiring the calibration points and no additional isotope labeled internal standards are required. The method was tested to identify the concentrations of 5mC and 5hmC in various sample types. The lowest level of DNA sample required in the case of 0.005% 5hmC is 0.5 µg.

Three Dimensional Cell Culturing for Modeling Adrenal and Pituitary Tumors.

In vitro monolayer conditions are not able to reproduce the complexity of solid tumors, still, there is scarce information about the 3D cell culture models of endocrine tumor types. Therefore, our aim was to develop in vitro 3D tumor models by different methodologies for adrenocortical carcinoma (H295R), pituitary neuroendocrine tumor (RC-4B/C and GH3) and pheochromocytoma (PC-12). Various methodologies were tested. Cell biological assays (cell viability, proliferation and live cell ratio) and steroid hormone production by HPLC-MS/MS method were applied to monitor cellular well-being. Cells in hanging drops and embedded in matrigel formed multicellular aggregates but they were difficult to handle and propagate for further experiments. The most widely used methods: ultra-low attachment plate (ULA) and spheroid inducing media (SFDM) were not the most viable 3D model of RC-4B/C and GH3 cells that would be suitable for further experiments. Combining spheroid generation with matrigel scaffold H295R 3D models were viable for 7 days, RC-4B/C and GH3 3D models for 7-10 days. ULA and SFDM 3D models of PC-12 cells could be used for further experiments up to 4 days. Higher steroid production in 3D models compared to conventional monolayer culture was detected. Endocrine tumor cells require extracellular matrix as scaffold for viable 3D models that can be one reason behind the lack of the usage of endocrine 3D cultures. Our models help understanding the pathogenesis of endocrine tumors and revealing potential biomarkers and therapeutic targets. They could also serve as an excellent platform for preclinical drug test screening.

Demethylation Status of Somatic DNA Extracted From Pituitary Neuroendocrine Tumors Indicates Proliferative Behavior.

BACKGROUND: Cytosine intermediaries 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), epigenetic hallmarks, have never been investigated in pituitary neuroendocrine tumors (PitNET). OBJECTIVE: To examine methylation-demethylation status of global deoxyribonucleic acid (DNA) in PitNET tissues and to assess its correlation with clinical and biological parameters. MATERIALS AND METHODS: Altogether, 57 PitNET and 25 corresponding plasma samples were collected. 5mC and 5hmC were investigated using liquid chromatography-tandem mass spectrometry. Expression of DNA methyltransferase 1 (DNMT1); tet methylcytosine dioxygenase 1 through 3 (TET1-3); and ubiquitin-like, containing PHD and RING finger domains 1 and 2 (UHRF1-2) were measured by reverse transcription-polymerase chain reaction. Levels of 5hmC and UHRF1-2 were explored by immunohistochemistry. Effect of demethylating agent decitabine was tested on pituitary cell lines. RESULTS: 5hmC/5mC ratio was higher in less differentiated PitNET samples. A negative correlation between Ki-67 proliferation index and 5hmC, 5hmC to 5mC ratio were revealed. Higher 5mC was observed in SF-1 + gonadotroph adenomas with a higher Ki-67 index. Expressions of TET2 and TET3 were significantly higher in adenomas with higher proliferation rate. UHRF1 showed gradually increased expression in higher proliferative adenoma samples, and a significant positive correlation was detected between UHRF2 expression and 5hmC level. Decitabine treatment significantly decreased 5mC and increased 5hmC levels in both cell lines, accompanied with decreased cell viability and proliferation. CONCLUSION: The demethylation process negatively correlated with proliferation rate and the ratio of 5hmC to 5mC was higher in less differentiated adenomas. Therefore, epigenetic markers can be potential biomarkers for PitNET behavior. Altering the epigenome in adenoma cells by decitabine decreased proliferation, suggesting that this treatment might be a novel medical treatment for PitNET.

The SDHB Arg230His mutation causing familial paraganglioma alters glycolysis in a new Caenorhabditis elegans model.

The conserved B-subunit of succinate dehydrogenase (SDH) participates in the tricarboxylic acid cycle (TCA) cycle and mitochondrial electron transport. The Arg230His mutation in SDHB causes heritable pheochromocytoma/paraganglioma (PPGL). In Caenorhabditiselegans, we generated an in vivo PPGL model (SDHB-1 Arg244His; equivalent to human Arg230His), which manifests delayed development, shortened lifespan, attenuated ATP production and reduced mitochondrial number. Although succinate is elevated in both missense and null sdhb-1(gk165) mutants, transcriptomic comparison suggests very different causal mechanisms that are supported by metabolic analysis, whereby only Arg244His (not null) worms demonstrate elevated lactate/pyruvate levels, pointing to a missense-induced, Warburg-like aberrant glycolysis. In silico predictions of the SDHA-B dimer structure demonstrate that Arg230His modifies the catalytic cleft despite the latter's remoteness from the mutation site. We hypothesize that the Arg230His SDHB mutation rewires metabolism, reminiscent of metabolic reprogramming in cancer. Our tractable model provides a novel tool to investigate the metastatic propensity of this familial cancer and our approach could illuminate wider SDH pathology.This article has an associated First Person interview with the first author of the paper.

Glutaminases as a Novel Target for SDHB-Associated Pheochromocytomas/Paragangliomas.

Pheochromocytoma/paragangliomas (Pheo/PGL) are rare endocrine cancers with strong genetic background. Mutations in the SDHB subunit of succinate dehydrogenase (SDH) predispose patients to malignant disease with limited therapeutic options and poor prognosis. Using a host of cellular and molecular biology techniques in 2D and 3D cell culture formats we show that SDH inhibition had cell line specific biological and biochemical consequences. Based on our studies performed on PC12 (rat chromaffin cell line), Hela (human cervix epithelial cell line), and H295R (human adrenocortical cell line) cells, we demonstrated that chromaffin cells were not affected negatively by the inhibition of SDH either by siRNA directed against SDHB or treatment with SDH inhibitors (itaconate and atpenin A5). Cell viability and intracellular metabolite measurements pointed to the cell line specific consequences of SDH impairment and to the importance of glutamate metabolism in chromaffin cells. A significant increase in glutaminase-1 (GLS-1) expression after SDH impairment was observed in PC12 cells. GLS-1 inhibitor BPTES was capable of significantly decreasing proliferation of SDH impaired PC12 cells. Glutaminase-1 and SDHB expressions were tested in 35 Pheo/PGL tumor tissues. Expression of GLS1 was higher in the SDHB low expressed group compared to SDHB high expressed tumors. Our data suggest that the SDH-associated malignant potential of Pheo/PGL is strongly dependent on GLS-1 expression and glutaminases may be novel targets for therapy.

The comprehensive characterization of adrenocortical steroidogenesis using two-dimensional ultra-performance liquid chromatography - electrospray ionization tandem mass spectrometry.

The perturbation of the homeostasis of adrenocortical steroids plays a fundamental role in several pathological conditions. Currently, only a few of the substances involved in steroidogenesis are routinely analysed in clinical laboratories for the diagnosis of these conditions. Recently, interest has grown over the development of clinical assays of endogenous steroids using liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, no approaches have assessed the adrenocortical steroidogenesis comprehensively. Here, a novel LC-MS/MS assay is presented for evaluating the serum levels of all respective major substances (aldosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, 11-deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol, dihydrotestosterone, 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, corticosterone, cortisol, cortisone, pregnenolone, progesterone and testosterone). The analysis time was 5.5 min following highly efficient solid phase extraction conducted on a novel polymer phase with N-polyvinylpyrrolidine branches. The method was validated in accordance with the respective guideline of the European Medicines Agency. The cross-validation of 8 analytes with immunoassays was also accomplished. Two-dimensional chromatography allowed the elution of the 16 analytes between 2.3-4.6 min and with a sufficient resolution of isobaric compounds. Quantitation was performed throughout the clinically relevant concentration ranges. Within-run accuracy was 87.1-115%, 90.0-109%, 87.2-111% and 87.6-107% at spiking levels 1 thru 4, while the precision was 4.7-27.9%, 2.9-17.7%, 5.6-13.9% and 1.9-15.0%, respectively. Between-run accuracy was 81.0-119.5, 85.2-113, 87.4-113 and 93.1-113%, respectively, while the precision was 3.4-13.5%, 2.0-10.2%, 2.1-15.0%, and 1.5-6.6%, respectively. In cross-validation studies, the mean percentage differences ranged between -51.4% (dehydroepiandrosterone sulfate) and 17.5% (dehydroepiandrosterone). The approach allows the comprehensive characterization of the adrenocortical steroid homeostasis in clinical diagnostics.

Simultaneous determination of thirteen different steroid hormones using micro UHPLC-MS/MS with on-line SPE system.

Ultratrace analysis of sample components requires excellent analytical performance in terms of limits of quantitation (LOQ). Micro UHPLC coupled to sensitive tandem mass spectrometry provides state of the art solution for such analytical problems. Using on-line SPE with column switching on a micro UHPLC-MS/MS system allowed to decrease LOQ without any complex sample preparation protocol. The presented method is capable of reaching satisfactory low LOQ values for analysis of thirteen different steroid molecules from human plasma without the most commonly used off-line SPE or compound derivatization. Steroids were determined by using two simple sample preparation methods, based on lower and higher plasma steroid concentrations. In the first method, higher analyte concentrations were directly determined after protein precipitation with methanol. The organic phase obtained from the precipitation was diluted with water and directly injected into the LC-MS system. In the second method, low steroid levels were determined by concentrating the organic phase after steroid extraction. In this case, analytes were extracted with ethyl acetate and reconstituted in 90/10 water/acetonitrile following evaporation to dryness. This step provided much lower LOQs, outperforming previously published values. The method has been validated and subsequently applied to clinical laboratory measurement.

GABA, glutamine, glutamate oxidation and succinic semialdehyde dehydrogenase expression in human gliomas.

BACKGROUND: Bioenergetic characterisation of malignant tissues revealed that different tumour cells can catabolise multiple substrates as salvage pathways, in response to metabolic stress. Altered metabolism in gliomas has received a lot of attention, especially in relation to IDH mutations, and the associated oncometabolite D-2-hydroxyglutarate (2-HG) that impact on metabolism, epigenetics and redox status. Astrocytomas and oligodendrogliomas, collectively called diffuse gliomas, are derived from astrocytes and oligodendrocytes that are in metabolic symbiosis with neurons; astrocytes can catabolise neuron-derived glutamate and gamma-aminobutyric acid (GABA) for supporting and regulating neuronal functions. METHODS: Metabolic characteristics of human glioma cell models - including mitochondrial function, glycolytic pathway and energy substrate oxidation - in relation to IDH mutation status and after 2-HG incubation were studied to understand the Janus-faced role of IDH1 mutations in the progression of gliomas/astrocytomas. The metabolic and bioenergetic features were identified in glioma cells using wild-type and genetically engineered IDH1-mutant glioblastoma cell lines by metabolic analyses with Seahorse, protein expression studies and liquid chromatography-mass spectrometry. RESULTS: U251 glioma cells were characterised by high levels of glutamine, glutamate and GABA oxidation. Succinic semialdehyde dehydrogenase (SSADH) expression was correlated to GABA oxidation. GABA addition to glioma cells increased proliferation rates. Expression of mutated IDH1 and treatment with 2-HG reduced glutamine and GABA oxidation, diminished the pro-proliferative effect of GABA in SSADH expressing cells. SSADH protein overexpression was found in almost all studied human cases with no significant association between SSADH expression and clinicopathological parameters (e.g. IDH mutation). CONCLUSIONS: Our findings demonstrate that SSADH expression may participate in the oxidation and/or consumption of GABA in gliomas, furthermore, GABA oxidation capacity may contribute to proliferation and worse prognosis of gliomas. Moreover, IDH mutation and 2-HG production inhibit GABA oxidation in glioma cells. Based on these data, GABA oxidation and SSADH activity could be additional therapeutic targets in gliomas/glioblastomas.

Evaluation of the Analytical and Clinical Concordance of 25-Hydroxyvitamin D Levels in Dried Blood Spots, Dried Serum Spots, and Serum as Potential Biorepository Specimens.

CONTEXT: The promising perspective of storing dried blood spot (DBS) for evaluating 25-hydroxyvitamin D (25OHD) levels is increasingly being realized. While strong correlations have been demonstrated between 25OHD levels measured in DBS and in systemic serum samples in earlier works, the clinical concordance of the assay results has not been evaluated. Moreover, the utility of dried serum spot (DSS), a highly suitable matrix for sample archiving, has not been investigated in this respect. METHODS: 25-hydroxycholecalciferol and 25-hydroxyergocalciferol levels were established selectively in DBS (n = 73) and DSS (n = 67) specimens obtained from deidentified whole blood and serum using a previously validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. In addition, total 25OHD levels were determined in the serum samples using the LIAISON 25OH Total Vitamin D Assay (LIA, n = 73). The analytical and clinical performance of the three approaches was compared pairwise. RESULTS: Deming regression, Bland-Altman analysis, and concordance correlation coefficients consistently demonstrated the lack of analytical equivalence among the three result sets. The overall percentage agreement of the clinical classifications (hypovitaminosis or euvitaminosis) was moderate (67.1%-83.6%). The delivery of positive cases was decreasing significantly in the order LIA>DSS>DBS (p < 0.05). CONCLUSIONS: The approaches tested did not deliver equivalent outputs either in an analytical or a clinical context. Therefore, specific reference ranges must be established for each matrix to avoid false clinical evaluation. 25OHD can be quantified when assay results are scaled by a factor of 1.60-1.67. Considering the convenience and efficiency of the storage and processing of DSS, along with the difficulties of quantifying 25OHD in real-life DBS samples accurately, DSS is proposed as an alternative for the long-term archiving of specimens.