Effects of processing of tomato on bioavailability of phenolic compounds and inflammatory biomarkers related to atherosclerosis

1. Leukocyte adhesion molecule expression:
Peripheral 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.
1.1. MAb used to mark adhesion molecules:
1.1.1. Anti-CD11a (LFA-1) (Bender MedSystems Diagnostics, Vienna)
1.1.2. Anti-CD40L (Bender MedSystems Diagnostics, Vienna)
1.1.3. Anti-CD11b (Mac-1) (Bender MedSystems Diagnostics, Vienna)
1.1.4. Anti-Syalil Lewis (anti-CD15s) (Pharmingen, San Diego, CA)
1.1.5. Anti-CD49d (VLA-4) (Cytogmos)
1.2. MAb used to mark T-lymphocytes: anti-CD2 (Caltag Laboratories, Burlingame, CA)
1.2. MAb used to mark monocytes: anti-CD14 (Caltag Laboratories, Burlingame, CA)

2. Soluble adhesion molecules:
The following serum soluble adhesion molecules (1-4) and other molecules (5-7) will be determined by enzyme-linked immunosorbent assay (ELISA) kits (Immunotech):
2.1. Soluble intercellular adhesion molecule-1 (sICAM-1)
2.2. Soluble vascular adhesion molecule 1 (sVCAM-1)
2.3. sE-selectin
2.4. sP-selectin
2.5. Soluble monocyte chemotactic protein-1 (sMCP-1)
2.6. Tumour necrosis factor-alpha (TNF-a)
2.7. Interleukin 1a (IL-1a)
3. Plasma and urine functional components study:
3.1. Plasma polyphenol concentration will be determined by Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS). The plasma polyphenol determinations will be carried out at 8 points during the 24h study, and urine polyphenol determinations at 0-6, 6-12 and 12-24 hours periods with the objective to obtain the plasma and urine phenolics kinetic and to investigate the different kinetic parameters used to evaluate their bioavailability; Area under the Curve (AUC), maximum concentration (Cmax), time to maximum plasma concentration (Tmax) and Time to maximum response (TMR).
3.2. Other studies to be carried out on the plasma and urine samples include:
3.2.1. Antioxidant capacity (Trolox-Equivalent Antioxidant Capacity [TEAC] assay, Oxygen Radical Absorbance Capacity [ORAC] assay)
3.2.2. Total phenolic concentration (Folin-Ciocalteu method)
3.2.3. Total Radical-trapping Antioxidant Parameter (TRAP) assay
3.2.4. Ferric Reducing Antioxidant Power (FRAP) assay

All variables (primary and secondary outcomes) will be measured at baseline and after each intervention.

1. Medical record:
1.1. A complete medical record will be obtained from all participants, which includes data on tomato intake, smoking and dietary habits.
1.2. Blood pressure and heart rate will be measured with an electronic apparatus Omron HEM-705CP (Netherlands).

2. Nutrition assessment and general analyses:
2.1. 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.
2.2. Overall nutrition will be determined by percentage of ideal weight, lean body mass and body mass index.
2.3. Waist perimeter will be measured.
2.4. The following measurements will also be obtained:
2.4.1. Red blood cell count
2.4.2. Haematocrit
2.4.3. Mean corpuscular volume
2.4.4. Leukocyte count
2.4.5. Glucose
2.4.6. Creatinine
2.4.7. Electrolytes
2.4.8. Uric acid
2.4.9. Transaminases
2.4.10. Lactate dehydrogenase
2.4.11. Alkaline phosphatase
2.4.12. Gamma-glutamyl transpeptidase
2.4.13. Bilirubin

3. Coagulation tests:
3.1. Platelet count
3.2. Prothrombin time
3.3. Plasma fibrinogen

4. Serum lipoprotein levels and others
4.1. Total cholesterol
4.2. Triglycerides
4.3. HDL cholesterol (cHDL)
4.4. cLDL
4.5. Apo A1
4.6. Apo B

5. Diet and exercise monitoring:
Monitoring of the diet and physical exercise will be carried out before and after each intervention.
5.1. 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. The foods ingested will be converted into nutritional values with the aid of the Professional Diet Balancer software (Cardinal Health Systems, Inc., Edina, MN).
5.2. Physical activity will also be evaluated with the Minnesota Leisure Time Physical Activity questionnaire which has also been validated in Spain.

All variables (primary and secondary outcomes) will be measured at baseline and after each intervention.