Metabolic fingerprinting of biological tissues has become an important area of research, particularly in the biomarker discovery field. Methods have inherent analytical variation, and new approaches are necessary to ensure that the vast numbers of intact metabolites present in biofluids are detected. Here, we describe an in-vial dual extraction (IVDE) method and a direct injection method that shows the total number of features recovered to be over 4500 from a single 20 μL plasma aliquot. By applying a one-step extraction consisting of a lipophilic and hydrophilic layer within a single vial insert, we showed that analytical variation was decreased. This was achieved by reducing sample preparation stages including procedures of drying and transfers. The two phases in the vial, upper and lower, underwent HPLC-QTOF analysis on individually customized LC gradients in both positive and negative ionization modes. A 60 min lipid profiling HPLC-QTOF method for the lipophilic phase was specifically developed, enabling the separation and putative identification of fatty acids, glycerolipids, glycerophospholipids, sphingolipids, and sterols. The aqueous phase of the extract underwent direct injection onto a 45 min gradient, enabling the detection of both polarities. The IVDE method was compared to two traditional extraction methods. The first method was a two-step ether evaporation and IPA resuspension, and the second method was a methanol precipitation typically used in fingerprinting studies. The IVDE provided a 378% increase in reproducible features when compared to evaporation and a 269% increase when compared to the precipitate and inject method. As a proof of concept, the method was applied to an animal model of diabetes. A 2-fold increase in discriminant metabolites was found when comparing diabetic and control rats with IVDE. These discriminant metabolites accounted for around 600 entities, out of which 388 were identified in available databases.

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Aims: A variety of vessels, such as resistance pulmonary arteries (PA) and fetoplacental arteries and the ductus arteriosus (DA) are specialized in sensing and responding to changes in oxygen tension. Despite opposite stimuli, normoxic DA contraction and hypoxic fetoplacental and PA vasoconstriction share some mechanistic features. Activation of neutral sphingomyelinase (nSMase) and subsequent ceramide production has been involved in hypoxic pulmonary vasoconstriction (HPV). Herein we aimed to study the possible role of nSMase-derived ceramide as a common factor in the acute oxygen-sensing function of specialized vascular tissues. Results: The nSMase inhibitor GW4869 and an anticeramide antibody reduced the hypoxic vasoconstriction in chicken PA and chorioallantoic arteries (CA) and the normoxic contraction of chicken DA. Incubation with interference RNA targeted to SMPD3 also inhibited HPV. Moreover, ceramide and reactive oxygen species production were increased by hypoxia in PA and by normoxia in DA. Either bacterial sphingomyelinase or ceramide mimicked the contractile responses of hypoxia in PA and CA and those of normoxia in the DA. Furthermore, ceramide inhibited voltage-gated potassium currents present in smooth muscle cells from PA and DA. Finally, the role of nSMase in acute oxygen sensing was also observed in human PA and DA. Innovation: These data provide evidence for the proposal that nSMasederived ceramide is a critical player in acute oxygen-sensing in specialized vascular tissues. Conclusion: Our results indicate that an increase in ceramide generation is involved in the vasoconstrictor responses induced by two opposite stimuli, such as hypoxia (in PA and CA) and normoxia (in DA). Antioxid. Redox Signal. 20, 1-14.

Abdominal aortic aneurysm (AAA) is perma-nent and localized dilation of the abdominal aorta. Intraluminal thrombus (ILT) is involved in evolution and rupture of AAA. Complex biological processes associated with AAA include oxidative stress, proteolysis, neovascularization, aortic inflammation, cell death, and extracellular matrix breakdown. Biomarkers of growth and AAA rupture could give a more nuanced indication for surgery, unveil novel pathogenic pathways, and open possibilities for pharmacological inhibition of growth. Differential analysis of metabolites released by normal and pathological arteries in culture may help to find molecules that have a high probability of later being found in plasma and start signaling processes or be useful diagnostic/prognostic markers. We used a LC-QTOF-MS metabolomic approach to analyze metabolites released by human ILT (divided into luminal and abluminal layers), aneurysm wall (AW), and healthy wall (HW). Statistical analysis was used to compare luminal with abluminal ILT layer, ILT with AW, and AW with HW to select the metabolites exchanged between tissue and external medium. Identified compounds are related to inflammation and oxidative stress and indicate the possible role of fatty acid amides in AAA. Some metabolites (e.g., hippuric acid) had not been previously associated to aneurysm, others (fatty acid amides) have arisen, indicating a very promising line of research.

The rat treated with streptozotocin has been proposed as the most appropriate model of systemic oxidative stress for studying antioxidant therapies. In that sense, rosemary extracts have long been recognized as having antioxidant properties, and folic acid may be able to improve endothelial progenitor cell function. A mixture containing both has been tested as a possible nutraceutical to improve health complications in diabetes. We have developed the methodology to evaluate metabolic changes in the urine of streptozotocin-induced diabetic rats after supplementing their diet with rosemary extract obtained with supercritical fluids (SFE) containing 10% folic acid in an acute but short-term study. It has been done with a metabolomics approach using LC-QTOF as an analytical tool. About 20 endogenous metabolites have been identified by databases and MS/MS showing statistically significant changes. Among them, several amino acids and their metabolites point to changes due to the effect of the gut microbiota. In addition, the comparison between control and streptozotocin-diabetic rats has permitted the showing of some metabolic coincidences between type 1 diabetes and other (possible) autoimmune diseases such as autism and/or Crohn's disease, and the nutraceutical intervention has succeeded in inducing changes in such biomarkers.