(1) The cardio-reno-metabolic benefits of the SGLT2 inhibitors canagliflozin (cana), dapagliflozin (dapa), ertugliflozin (ertu), and empagliflozin (empa) have been demonstrated, but it remains unclear whether they exert different off-target effects influencing clinical profiles. (2) We aimed to investigate the effects of SGLT2 inhibitors on mitochondrial function, cellular glucose-uptake (GU), and metabolic pathways in human-umbilical-vein endothelial cells (HUVECs). (3) At 100 µM (supra-pharmacological concentration), cana decreased ECAR by 45% and inhibited GU (IC5o: 14 µM). At 100 µM and 10 µM (pharmacological concentration), cana increased the ADP/ATP ratio, whereas dapa and ertu (3, 10 µM, about 10× the pharmacological concentration) showed no effect. Cana (100 µM) decreased the oxygen consumption rate (OCR) by 60%, while dapa decreased it by 7%, and ertu and empa (all 100 µM) had no significant effect. Cana (100 µM) inhibited GLUT1, but did not significantly affect GLUTs’ expression levels. Cana (100 µM) treatment reduced glycolysis, elevated the amino acids supplying the tricarboxylic-acid cycle, and significantly increased purine/pyrimidine-pathway metabolites, in contrast to dapa (3 µM) and ertu (10 µM). (4) The results confirmed cana´s inhibition of mitochondrial activity and GU at supra-pharmacological and pharmacological concentrations, whereas the dapa, ertu, and empa did not show effects even at supra-pharmacological concentrations. At supra-pharmacological concentrations, cana (but not dapa or ertu) affected multiple cellular pathways and inhibited GLUT1.

Rationale: Pulmonary hypertension (PH) poses a significant health threat. Current biomarkers for PH lack specificity and have poor prognostic capabilities. Objectives: To develop better biomarkers for PH that are useful for patient identification and management. Methods: An explorative analysis was conducted of a broad spectrum of metabolites in patients with PH, healthy control subjects, and diseased control subjects in training and validation cohorts, together with in vitro studies on human pulmonary arteries. Measurements and Main Results: High-resolution mass spectrometry was performed in 233 subjects coupled with machine learning analysis. Histologic and gene expression analysis was conducted, with a focus on lipid metabolism in human pulmonary arteries of idiopathic pulmonary arterial hypertension lungs and assessment of the acute effects of extrinsic fatty acids (FAs). We enrolled a training cohort of 74 patients with PH, 30 diseased control subjects without PH, and 65 healthy control subjects, as well as an independent validation cohort of 64 subjects. Among other metabolites, FAs were significantly increased. Machine learning showed a high diagnostic potential for PH. In addition, we developed fully explainable lipid ratios with exceptional diagnostic accuracy for PH (areas under the curve of 0.89 in the training cohort and 0.90 in the external validation cohort), outperforming machine learning results. These ratios were also prognostic and complemented established clinical markers and scores, significantly increasing their hazard ratios for mortality risk. Idiopathic pulmonary arterial hypertension lungs showed lipid accumulation and altered expression of lipid homeostasis–related genes. In human pulmonary artery smooth muscle and endothelial cells, FAs caused excessive proliferation and barrier dysfunction, respectively. Conclusions: Our metabolomics approach suggests that lipid alterations in PH provide diagnostic and prognostic information, complementing established markers. These alterations may reflect pathologic changes in the pulmonary arteries of patients with PH.

Timely centrifugation of blood for plasma preparation is a key step to ensure high plasma quality for analytics. Delays during preparation can significantly influence readouts of key clinical parameters. However, in a routine clinical environment, a strictly controlled timeline is often not feasible. The next best approach is to control for sample preparation delays by a marker that provides a readout of the time-dependent degradation of the sample. In this study, we explored the usefulness of glutathione status as potential marker of plasma preparation delay. As the concentration of glutathione in erythrocytes is at least two orders of magnitude higher than in plasma, even the slightest leakage of glutathione from the cells can be readily observed. Over the 3 h observation period employed in this study, we observed a linear increase of plasma concentrations of both reduced (GSH) and oxidized glutathione (GSSG). Artificial oxidation of GSH is prevented by rapid alkylation with N-ethylmaleimide directly in the blood sampling vessel as recently published. The observed relative leakage of GSH was significantly higher than that of GSSG. A direct comparison with plasma lactate dehydrogenase activity, a widely employed hemolysis marker, clearly demonstrated the superiority of our approach for quality control. Moreover, we show that the addition of the thiol alkylating reagent NEM directly to the blood tubes does not influence downstream analysis of other clinical parameters. In conclusion, we report that GSH gives an excellent readout of the duration of plasma preparation and the associated pre-analytical errors.

Background: We aimed to gain insights in a co-culture of 10 bacteria and their postbiotic supernatant. Methods: Abundances and gene expression were monitored by shotgun analysis. The supernatant was characterized by liquid chromatography mass spectroscopy (LC-MS) and gas chromatography mass spectroscopy (GC-MS). Supernatant was harvested after 48 h (S48) and 196 h (S196). Susceptibility testing included nine bacteria and C. albicans. Bagg albino (BALBc) mice were fed with supernatant or culture medium. Fecal samples were obtained for 16S analysis. Results: A time-dependent decrease of the relative abundances and gene expression of L. salivarius, L. paracasei, E. faecium and B. longum/lactis and an increase of L. plantarum were observed. Substances in LC-MS were predominantly allocated to groups amino acids/peptides/metabolites and nucleotides/metabolites, relating to gene expression. Fumaric, panthotenic, 9,3-methyl-2-oxovaleric, malic and aspartic acid, cytidine monophosphate, orotidine, phosphoserine, creatine, tryptophan correlated to culture time. Supernatant had no effect against anaerobic bacteria. S48 was reactive against S. epidermidis, L. monocytogenes, P. aeruginosae, E. faecium and C. albicans. S196 against S. epidermidis and Str. agalactiae. In vivo S48/S196 had no effect on alpha/beta diversity. Linear discriminant analysis effect size (LEfSe) and analysis of composition of microbiomes (ANCOM) revealed an increase of Anaeroplasma and Faecalibacterium prausnitzii. Conclusions: The postbiotic supernatant had positive antibacterial and antifungal effects in vitro and promoted the growth of distinct bacteria in vivo.