Microbiota modulates the steroid response to acute immune stress in male mice.

Microbiota plays a role in shaping the HPA-axis response to psychological stressors. To examine the role of microbiota in response to acute immune stressor, we stimulated the adaptive immune system by anti-CD3 antibody injection and investigated the expression of adrenal steroidogenic enzymes and profiling of plasma corticosteroids and their metabolites in specific pathogen-free (SPF) and germ-free (GF) mice. Using UHPLC-MS/MS, we showed that 4 hours after immune challenge the plasma levels of pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone (CORT), 11-dehydroCORT and their 3α/ß-, 5α-, and 20α-reduced metabolites were increased in SPF mice, but in their GF counterparts, only CORT was increased. Neither immune stress nor microbiota changed the mRNA and protein levels of enzymes of adrenal steroidogenesis. In contrast, immune stress resulted in downregulated expression of steroidogenic genes (Star, Cyp11a1, Hsd3b1, Hsd3b6) and upregulated expression of genes of the 3α-hydroxysteroid oxidoreductase pathway (Akr1c21, Dhrs9) in the testes of SPF mice. In the liver, immune stress downregulated the expression of genes encoding enzymes with 3ß-hydroxysteroid dehydrogenase (HSD) (Hsd3b2, Hsd3b3, Hsd3b4, Hsd3b5), 3α-HSD (Akr1c14), 20α-HSD (Akr1c6, Hsd17b1, Hsd17b2) and 5α-reductase (Srd5a1) activities, except for Dhrs9, which was upregulated. In the colon, microbiota downregulated Cyp11a1 and modulated the response of Hsd11b1 and Hsd11b2 expression to immune stress. These data underline the role of microbiota in shaping the response to immune stressor. Microbiota modulates the stress-induced increase in C21 steroids, including those that are neuroactive that could play a role in alteration of HPA axis response to stress in GF animals.

Columns in analytical-scale supercritical fluid chromatography: From traditional to unconventional chemistries.

Within this review, we thoroughly explored supercritical fluid chromatography (SFC) columns used across > 3000 papers published from the first study carried out under SFC conditions in 1962 to the end of 2022. We focused on the open tubular capillary, packed capillary, and packed columns, their chemistries, dimensions, and trends in used stationary phases with correlation to their specific interactions, advantages, drawbacks, used instrumentation, and application field. Since the 1990s, packed columns with liquid chromatography and SFC-dedicated stationary phases for chiral and achiral separation are predominantly used. These stationary phases are based on silica support modified with a wide range of chemical moieties. Moreover, numerous unconventional stationary phases were evaluated, including porous graphitic carbon, titania, zirconia, alumina, liquid crystals, and ionic liquids. The applications of unconventional stationary phases are described in detail as they bring essential findings required for further development of the supercritical fluid chromatography technique.

Fast Optimization of Supercritical Fluid Chromatography-Mass Spectrometry Interfacing Using Prediction Equations.

The effect of makeup solvent composition in ultrahigh-performance supercritical fluid chromatography-triple quadrupole mass spectrometry using electrospray ionization was studied using a set of 91 compounds, 3 stationary phases, and 2 organic modifiers of the mobile phase. The 24 tested makeup solvents included pure alcohols and methanol in combination with commonly used additives such as water, formic and acetic acid, ammonia, and ammonia salts with varying molarity. The behavioral trends for different makeup solvent additives were established in the first step. Subsequently, the correlations between physicochemical properties and the MS responses were calculated using the Pearson correlation test and matrix plots. The regression analysis was performed using five descriptors: molecular weight, pKa, log P, number of hydrogen donors/acceptors, and the MS responses obtained with methanol as the makeup solvent. The resulting regression equations had a high prediction rate calculated as R2-predicted coefficient, especially when 10 mmol/L ammonium in methanol was used as an organic modifier of the mobile phase in positive mode. The trueness of these equations was tested via the comparison between experimental and predicted responses expressed as R2. Values of R2 > 0.8 were found for 88% of the proposed equations. Thus, the MS response could be measured using only one makeup solvent and the responses of other makeup solvents could be easily estimated. The suitability and applicability of determined regression equations was confirmed by the analysis of 13 blind probes, i.e., compounds not included in the original set of analytes. Moreover, the predicted and experimental responses followed the same increasing/decreasing trend enabling one to predict makeup solvent compositions leading to the highest sensitivity.

Characterization of glycosphingolipids from gastrointestinal stromal tumours.

Gastrointestinal stromal tumours (GISTs) are the major nonepithelial neoplasms of the human gastrointestinal tract with a worldwide incidence between 11 and 15 per million cases annually. In this study the acid and non-acid glycosphingolipids of three GISTs were characterized using a combination of thin-layer chromatography, chemical staining, binding of carbohydrate recognizing ligands, and mass spectrometry. In the non-acid glycosphingolipid fractions of the tumors globotetraosylceramide, neolactotetraosylceramide, and glycosphingolipids with terminal blood group A, B, H, Lex, Lea, Ley and Leb determinants were found. The relative amounts of these non-acid compounds were different in the three tumour samples. The acid glycosphingolipid fractions had sulfatide, and the gangliosides GM3, GD3, GM1, Neu5Acα3neolactotetraosylceramide, GD1a, GT1b and GQ1b. In summary, we have characterized the glycosphingolipids of GISTs and found that the pattern differs in tumours from different individuals. This detailed characterization of glycosphingolipid composition of GISTs could contribute to recognition of new molecular targets for GIST treatment and sub-classification.