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The differentiation of endometrial stromal cells, named decidualization, is essential for the proper formation of the materno-fetal interphase. One important feature of decidualization is the increased glucose consumption and its utilization by endometrial cells to produce energy. Besides glucose, fatty acids are another important energy source for living cells and it has been described that endometrial stromal cells rely on the proper function of the oxidation of fatty acids for the correct decidualization. It is, however, unknown whether the turn-over of fatty acid degradation is modified during decidualization. Furthermore, it is also unknown how the final products of glucose and fatty acid catabolism are related to the function of the tricarboxylic acid cycle for the efficient ATP production. In this study, we evaluated the content levels of different intermediate metabolites and the expression of the key enzymes related to the degradation of glucose and fatty acids during the in vitro decidualization of human endometrial stromal cells. Our results suggest that human endometrial stromal cells undergo energetic metabolic changes during decidualization and that decidualizing and non-decidualizing cells differ in the level of activation of different metabolic pathways and, probably, in the use of intermediate metabolites.

BACKGROUND AND PURPOSE Trimethylamine-N-oxide (TMAO) is a biomarker of the gut microbiome and correlates with the risk of cardiovascular diseases. However, conflicting data exist on the specific role of TMAO in ischaemic stroke patients. We aimed to analyze the time course of TMAO levels in stroke patients compared with controls. METHODS In this prospective, case-control study, patients suffering from ischaemic stroke (onset

Purpose Endometrial carcinoma is the second most common gynecological malignancy. Until today lacking a screening tool. A blood-based biomarker could help address this need. Methods The expression levels of 30 acylcarnitines, 18 amino acids, 6 miRNAs, and 7 DNA methylation sites were measured in blood samples from 331 women (20 EC, 14 benign uterine lesions (benign), 140 breast cancers (BC), 157 controls). Areas under the ROC curves (AUC), sensitivity (sens.) and specificity (spec.) were computed to identify the variables best distinguishing. Results The best top ten markers for the four comparisons (cancer vs. cancer-free; EC vs. BC, EC vs. controls; EC vs. benign), were identified via AUC. Malonylcarnitine distinguished best patients with EC from controls (AUC: 0.827, sens. 80%, spec. 73.1%) or BC (AUC: 0.819, sens. 84.3%, spec. 80%) being most notable. Tryptophan best differentiated benign from EC (AUC: 0.846, sens. 70%, spec. 92.9%). Conclusions The levels of the analyzed blood markers yielded promising results in the detection of EC and warrant further evaluation.

Introduction: Trimethylamine N-oxide (TMAO) derived from dietary sources has been linked to a higher risk of atherosclerotic disease. However, the underlying mechanisms are not fully understood. Impairment of endothelial cells (ECs) by chronic inflammation or metabolic cues can lead to a loss of EC phenotype and function towards a mesenchymal cell type, a process contributing to atherosclerosis termed endothelial-to-mesenchymal transition (EndMT). Here, we investigate the potential uptake of TMAO and its impact on endothelial inflammation and EndMT. Methods & Results: Using tandem mass spectrometry, we found ECs to take up TMAO in a time-dependent manner (0.5 nmol/mg within 30 min rising to 5.8 nmol/mg TMAO/total protein after 24 h). The induction of ICAM1 (+26.5-fold) and E-Selectin (+77.7-fold, both p The multitude of known human solute carrier proteins were screened in ECs for identification of a potential TMAO transporter. Single-cell RNA-sequencing revealed the Endothelial TMAO Transporter 1 (ETT1) to be upregulated in inflammatory-activated ECs of the myocardium. Overexpression of ETT1 resulted in a 61.7% higher intracellular TMAO concentration in ECs after 30 min of treatment. Downregulation of ETT1 by siRNA prevented TMAO-induced inflammation (-112% induction of E-Selectin and -105% induction of ICAM1) and EndMT (-315% induction of SM22 and -533% induction of Calponin 1) and restored EC barrier function (-78% induction of permeability). Conclusions: In summary, our study identifies that TMAO uptake into human ECs causes increased inflammation and induces EndMT. The TMAO-mediated impairment of EC function is prevented by down-regulation of the newly identified TMAO transporter ETT1. Our findings may help to better understand the interaction of diet and atherosclerosis.

Monitoring phenylalanine (Phe) concentrations is critical for the management of phenylketonuria (PKU). This can be done in dried blood spots (DBS) or in EDTA plasma derived from capillary or venous blood. Different techniques are used to measure Phe, the most common being flow-injection analysis tandem mass spectrometry (FIA-MS-MS) and ion exchange chromatography (IEC). Significant differences have been reported between Phe concentrations in various sample types measured by different techniques, the cause of which is not yet understood. We measured Phe concentrations in 240 venous blood samples from 199 patients with hyperphenylalaninemia in dried blood spots, EDTA plasma and erythrocytes by FIA-MS-MS and IEC. Phe concentrations were significantly lower in erythrocytes than in plasma leading to about 19% lower Phe DBS concentrations compared with plasma independent from the method used for quantification. As most therapy recommendations for PKU patients are based on plasma concentrations reliable conversion of DBS into plasma concentrations is necessary. Variances of Phe concentrations in plasma and DBS are not linear but increases with higher concentrations indicating heteroscedasticity. We therefore suggest the slope of the 75th percentile from quantile regression as a correction factor.

Study question Is the activity of the β-oxidation pathway, involved in the degradation of fatty acids, modified during in vitro decidualization of human endometrial stromal cells (HESC)? Summary answer The level of expression of fatty acid´s transporters suggests that the activity of the mitochondrial β-oxidation pathway is increased during in vitro decidualization of HESC. What is known already The differentiation of endometrial stromal cells (ESC), named decidualization, is essential for the proper formation of the materno-fetal interphase. One important feature of decidualization is the increased glucose consumption. In the endometrium, glucose is incorporated into ESC by glucose-transporters (GLUT). Fatty acids are another important energy source in living cells. Fatty acids are transported into mitochondria by the carnitine-palmitoyl-transferases 1 and 2 (CPT1 and 2) and are degraded there through the β-oxidation pathway. It has been described that the inhibition of CPT1 affects ESC decidualization. However, it is unknown whether the turn-over of fatty acids degradation is modified during decidualization. Study design, size, duration This study was performed using primary HESC. Endometrial biopsies (mid-late proliferative-phase) were obtained from healthy-regularly-cycling women (33.6±2.2 years-old) after written informed consent was obtained (protocol approved by Ethics committee no. S-239/2005). HESC were decidualized (D) in vitro with a decidualization-cocktail (containing: medroxyprogesterone acetate, estradiol and 8-Bromo-cyclic adenosine monophosphate) for 6 days. Non-decidualized (ND) controls were treated with vehicle solutions. Cell-culture supernatant and cell extracts were collected for the evaluation of protein/gene expression and metabolite content. Participants/materials, setting, methods Decidualization was evaluated by measuring prolactin (PRL) protein levels in cell-culture supernatant (mU/l). Changes in mRNA expression levels of GLUT1, CPT1A and CPT2 were evaluated by real-time polymerase chain reaction (RT-PCR). Analysis was performed by the ΔΔCt method (internal control: RPLP0) (fold change -FC- in D compared to ND cells). Contents of acylcarnitines were evaluated by Electrospray Ionization-Tandem Mass Spectrometry (ESI-MS/MS) (nmol/mg of total protein). N = 5, mean±SEM. Paired Student’s t-test was used for statistical analysis. Main results and the role of chance PRL protein levels in cell-culture supernatant were significative increased in HESC treated with the decidualization-cocktail compared to ND cells (ND 16.80±0.73 mU/l; D 684.20±219.80 mU/l, *p 0,05). Limitations, reasons for caution This study was performed in vitro using primary HESC treated with a decidualization-cocktail. The interconnection of different metabolic pathways within a living cell is very complex. Further studies are necessary to define whether the different intermediate metabolites of the mitochondrial β-oxidation pathway are being used by related-metabolic pathways during decidualization. Wider implications of the findings The regulation of the energy metabolism and its interconnection with other important intra-cellular metabolic pathways is of great importance for cellular function. Our results contribute to highlight the importance of the regulation of fatty acids degradation during decidualization. Further insights into HESC metabolism could facilitate the improvement of womeńs health. Trial registration number not applicable

Introduction: Trimethylamine-N-oxide (TMAO ) is a biomarker of the gut microbiome and has been reported to be associated with cardiovascular diseases. However, there have been conflicting reports regarding its association with ischemic stroke. As TMAO values could be elevated before stroke or change after stroke we aimed at analyzing the early time course of TMAO values after acute ischemic stroke. Methods: In this prospective ongoing case-control study, we included patients with ischemic stroke within the first 24 hours after last-seen-well and control patients treated for other neurologic disorders with only few cardiovascular risk factors at a tertiary care university hospital. The TMAO-values on admission and after 48 hours were analyzed. Patients and controls were excluded if they were treated with antibiotics within the last 30 days and if they were treated with probiotics or chemotherapy. Results: 99 patients have been recruited so far (63 stroke patients and 36 controls). The median NIHSS score in stroke patients was 3 (interquartile range: 1-8). 89% of the stroke patients vs. 94% of the control group had a normal kidney function. Stroke patient had significantly higher median TMAO values compared to control patients: 5.4 (3.0-8.0) vs. 2.8 (1.6-5.0) μmol/l, p=0.002. Interestingly, there was a significant decrease in TMAO values in stroke patients from admission to 48 hours after the stroke (median decrease 0.7 (0.6-4.2) p=0.004), which was not found for the control group (median decrease 0.1 (-0.9-1.3), p=0.758). In consequence, there was no significant difference between the groups for the TMAO-values after 48 hours (3.4 (2.5-5.1) vs. 3.0 (1.6-4.5) μmol/l, p=0.081). Conclusion: This study emphasizes the importance of the TMAO time course after stroke. TMAO values decrease dramatically within the first days after stroke which makes it essential to standardize the time point of analysis to ensure comparability in future studies.

BACKGROUND AND AIMS:Inborn errors of purine and pyrimidine metabolism are a diverse group of disorders with possible serious or life-threatening symptoms. They may be associated with neurological symptoms, renal stone disease or immunodeficiency. However, the clinical presentation can be nonspecific and mild so that a number of cases may be missed. Previously published assays lacked detection of certain diagnostically important biomarkers, including SAICAr, AICAr, beta-ureidoisobutyric acid, 2,8-dihydroxyadenine and orotidine, necessitating the use of separate assays for their detection. Moreover, the limited sensitivity for some analytes in earlier assays may have hampered the reliable detection of mild cases. Therefore, we aimed to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that allows the simultaneous and sensitive detection of an extended range of purine and pyrimidine biomarkers in urine. METHODS:The assay was developed and validated using LC-MS/MS and clinically tested by analyzing ERNDIM Diagnostic Proficiency Testing (DPT) samples and further specimens from patients with various purine and pyrimidine disorders. RESULTS:Reliable determination of 27 analytes including SAICAr, AICAr, beta-ureidoisobutyric acid, 2,8-dihydroxyadenine and orotidine was achieved in urine following a simple sample preparation. The method clearly distinguished pathological and normal samples and differentiated between purine and pyrimidine defects in all clinical specimens. CONCLUSIONS:A LC-MS/MS assay allowing the simultaneous, sensitive and reliable diagnosis of an extended range of purine and pyrimidine disorders has been developed. The validated method has successfully been tested using ERNDIM Diagnostic Proficiency Testing (DPT) samples and further clinical specimens from patients with various purine and pyrimidine disorders. Sample preparation is simple and assay duration is short, facilitating an easier inclusion of the assay into the diagnostic procedures.