Effects of acute and one month ingestion of biologically aged Andalusian wines on endothelial function

1. Leukocyte adhesion molecule expression
Lymphocyte and monocyte adhesion molecules on these cells will be marked with monoclonal antibodies (MAb) conjugated with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE) by direct double immunofluorescence. The MAb of the adhesion molecules used will be: anti-CD11a (LFA-1), anti-CD40L, anti-CD11b (Mac-1) (Bender MedSystems Diagnostics, Vienna), anti-Syalil Lewis (anti-CD15s) (Pharmingen, San Diego, CA), anti-CD49d (VLA-4) (Cytogmos) and CD31 (BD, NJ, USA). The monoclonal antibodies used to mark the T-lymphocytes will be anti-CD2 and monocytes, anti-CD14 (Caltag Laboratories, Burlingame, CA).
2. Soluble adhesion molecules
The following serum soluble adhesion molecules will be determined by ELISA kits:
2.1. sICAM-1, sVCAM-1, PECAM, hs-CRP, sE-selectin, and sP-selectin, as well as sMCP-1, TNF-a, IL-6 and IL-1B (Immunotech).
2.2. Nuclear Factor Kappa B by western blot of peripheral blood mononuclear cells.
2.3. Genes and proteins involved in inflammatory response will be determined by real time PCR and Western blot analysis (MCP-1, TF and TFPI, as markers of inflammation and LRP and the LDL receptor as lipoproteic receptors). Moreover, the expression metalloproteases and their activity will also be analyzed.
3. Oxidative stress
The following plasma molecules will be determined: superoxide dismutase (SOD) with the Superoxide Dismutase Assay SD125 (Randox Laboratories Ltd, UK), catalase (CAT) with the Catalase Assay (Cayman Chemical, Ann Arbor, MI, USA), glutathione reductase (GR) and reduced glutathione (GSH) with the Glutathione Reductase Assay GR2368 (Randox Laboratories Ltd, UK), glutation peroxidase with the RS 504 assay (Randox Laboratories Ltd, UK), el complejo GSH/GSSG (Bioxytech OxisResearch, Foster City, CA, USA), protein carbonyl groups with the Protein Carbonyl Assay (Cayman Chemical, Ann Arbor, MI, USA), lipid peroxides (LPO) with the LPO-CC Assay (Kamiya Biomedical Company, Seattle, USA), malondialdehyde (MDA) and 4-hydroxyalkenals (HAE) with the Bioxytech LPO-586 (Oxys Research, Portland, OR, USA), total antioxidant capacity with the Total Antioxidant Powder (aqueous) Assay (Neogen Corporation, Lexington, KY, USA) and hidroxideoxiguanosina 8-(8-OHdG) with the 8-OHdG Check ELISA Kit (Genox Corporation, Baltimore, USA) in plasma in addition to the concentration of urinary isoprostanes with the Urinary Isoprostane Assay (Neogen Corporation, Lexington, KY, USA).
4. Nitric oxide
The concentration of nitrate / nitrite (NO (x)) in plasma will be determined through purifying the samples with Microcon YM-10 filters (Micon Bioseparations, Millipore, Billerica, MA, USA) and the commercial kit Nitrate / Nitrite Colorimetric Assay Kit (Cayman Chemical, Ann Arbor, MI, USA). In addition, the concentration of nitric oxide synthase (NOS) in plasma will be determined with the commercial kit Bioxytech ® Nitric Oxide Synthase Assay Kit ™ (Oxys Research, Portland, OR, USA).
5. Wine biomarkers and urinary polyphenols
The urinary polyphenol levels will be also determined with the Folin-Ciocalteau assay (Sigma-Aldrich-Fluka, St. Louis, MO, USA). Furthermore, tartaric acid in urine as biomarker of wine intake will be also quantified by LC-MS/MS.

1. Medical record
A complete medical record will be obtained from all participants, which included data on alcohol intake, smoking and dietary habits. Blood pressure and heart rate will be measured with an electronic apparatus Omron HEM-705CP (Netherlands).
2. Nutrition assessment and general analyses
All participants will complete a validated nutritional questionnaire at baseline to determine the total quantity of calories ingested in the previous month as well as the proportion corresponding to carbohydrates, lipids and proteins by using the Food Processor Nutrition & Fitness software (esha RESEARCH, Salem, OR, USA) . Overall nutrition will be determined by percentage of ideal weight, lean body mass and body mass index. Waist Perimeter will be measured. The proteic nutrition will be determined on the basis of the following parameters: hemoglobin, total lymphocyte count, total proteins, albumin, prealbumin, transferrin and retinol-binding protein. Serum and intraerythrocytary folic acid concentrations will be measured, as well as serum vitamin A, B1, B12, C, E, B-carotenes, Zn, Mg and Se concentrations.
Moreover, the following measurements will also be obtained: red blood cell count, hematocrit, mean corpuscular volume, leukocyte count, glucose, creatinine, electrolytes, uric acid, transaminases, lactate dehydrogenase, alkaline phosphatase, gammaglutamyl transpeptidase and bilirrubin.
3. Coagulation tests
The following parameters will also be determined: platelet count, prothrombin time, and plasma fibrinogen.
4. Serum lipoproteins and others
Total cholesterol, triglycerides, cHDL, cLDL, Apo A1, Apo B, lipoprotein (a) and homocysteine will be determined.
5. Diet and exercise monitoring
All participants will follow an isocaloric diet prepared according to their personal preferences. The diet will be strictly monitored during the study. Diet compliance will be assessed from 7-days diet records administered before each evaluation. This assessment will be administered by trained personnel. The foods ingested will be converted into nutritional values with the aid of the Professional Diet Balancer software (Cardinal Health Systems, Inc., Edina, MN). Physical activity will also be evaluated with the Minnesota Leisure Time Physical Activity questionnaire which has also been validated in Spain. Control of the diet and physical exercise will be carried out before and after each intervention, the same day on which the clinical examinations are performed and blood is withdrawn for immunologic studies.