Onsumed two glasses of WW distributed in between two distinct meals equivalent to 270 mL of WW, 27 g of alcohol, 2.8 mg of TYR, and 0.4 mg of HT. WW TYR intervention consisted with the equivalent quantity of WW complemented with a capsule of 25 mg TYR. Hence, females consumed 25 mg of TYR and male participants consumed 50 mg of TYR. The study was performed in accordance using the Helsinki Declaration and approved by the regional Ethical Committee (CEIm-Parc de Salut Mar) and registered within the ClinicalTrials. gov (accessed on 20 October 2021) database (NCT02783989). Informed consent was obtained from all subjects involved inside the study. two.three. Cardiovascular Biomarker Assessments Endothelial function was measured in all participants at the starting and in the end of each and every intervention period, monitoring the reactive hyperemia index (RHI) plus the augmentation index (AI) measured with EndoPAT 2000 (Itamar Healthcare Inc., Caesarea, Israel). Biomarkers related with cardiovascular health like total cholesterol, HDL-c, antithrombin III (ATIII), and D-dimer (DD) have been measured by automated enzymatic solutions. LDL cholesterol was calculated by the Friedewald formula whenever triglycerides have been inferior to 300 mg/dL. Serum high-sensitivity C reactive protein (hs-CRP), ATIII, and DD were determined by immunoturbidimetry (Horiba, Montpellier, France; Spinreact, Girona, Spain). Homocysteine (Hcy) in plasma was measured by gas chromatography-mass spectrometry (GC-MS) immediately after liquid iquid extraction. Oxidized LDL (oxLDL), endothelin 1 (ET1), and plasminogen activator inhibitor-1 (PAI-1) were measured in plasma by ELISA (Mercodia, Uppsala, Sweden; Invitrogen, CA, USA; and Affymetrix, CA, USA, respectively). 2.four. Plasma Sample for Analysis Blood samples had been obtained at the starting and in the end of each and every intervention period at fasting situations. Venous blood samples were collected in tubes 5-Ethynyl-2′-deoxyuridine Autophagy containing EDTA and centrifuged at 1700g for 15 min at four C to obtain plasma. All samples had been stored at -80 C till evaluation. 2.5. Lipidomic Profile Evaluation Circulating levels of ceramides (Cer) and extra lipids (MAGs, DAGs, HexCers, LPCs, SM, and S1P) in human plasma have been determined by liquid chromatography coupled to a tandem mass spectrometry (LC S/MS) system, as previously described [18] with slight modifications. Briefly, ten of plasma sample was spiked with one hundred of methanolic ice-cold internal regular remedy containing a mixture of 11 deuterated compounds supplied by Avanti Polar Lipids (see Table A1 for further facts). An added one hundred of ice-cold methanolic resolution was added. Immediately after vortexing and centrifugation (five min,Antioxidants 2021, 10,four of3500 rpm, 4 C), the supernatant was transferred to an HPLC vial and five was injected into the LC-MS/MS program. The chromatographic separation with the lipid species was performed working with an Acquity UPLC instrument (Waters Associates, Milford, MA, USA) operated employing the MassLynx 4.1 computer β-Lapachone Cancer software. The LC system was equipped with an Acquity UPLC(BEH C18, 1.7 , two.1 one hundred mm) column (Waters Associates). The flow price was 0.3 mL/min plus the temperature on the column was set at 55 C. An isocratic process was used, using a resolution of 1 mM of ammonium formate (NH4 HCOO) and 0.01 HCOOH in methanol because the mobile phase solvent. The total run-time was 5 min. The detection with the ammonium adducts ([M NH4 ] in the case of diacylglycerols) and protonated adducts ([M H] for the other lipid species) was performed having a triple quadrupole (Xevo.