EVOO Coratina Intervention on Lipids in Non-diabetic Hypertensive Patients Undergoing Hemodialysis (EVOOC-3H)
EVOO Coratina Intervention on Lipids in Non-diabetic Hypertensive Patients Undergoing Hemodialysis (EVOOC-3H)
Twenty-four non-diabetic hypertensive and hyperlipidemic patients undergoing periodic chronic
 hemodialysis will be enrolled for receiving extravirgin olive oil (EVOO) Coratina (12
 patients) or refined olive oil (12 patients). Aim of the study is to evaluate the effect of
 EVOO-C on serum lipid levels. Randomization will be done centrally with appropriate
 stratification. Sample size is opportunistic because this is a pilot study. Dietary and
 clinical monitoring will be done by nephrologists, cardiologists and dieticians.
Detailed Description: EXTRAVIRGIN OLIVE OIL CORATINA INTERVENTION ON LIPIDS IN NON-DIABETIC
 HYPERTENSIVE (H) PATIENTS WITH HYPERLIPIDEMIA (H)UNDERGOING PERIODIC HEMODIALYSIS (H)
 (EVOOC-3H) INTRODUCTION What is known. The primary causes of death in patients undergoing
 chronic hemodialysis (HD) are the cardiovascular events.

 Cardiovascular risk factors(hypertension, type 2 diabetes, obesity, high levels of low
 density lipoprotein (LDL) , low levels of high density lipoprotein cholesterol (HDL), smoking
 and reduced physical activity) are almost universally present in people on HD. Evidence from
 randomized clinical trials demonstrate that statin administration in patients undergoing HD
 does not reduce the risk of adverse vascular outcomes. In the general population, there is
 growing evidence that the Mediterranean diet supplemented with olive oil implies
 cardiovascular and survival advantage. The favorable effects of the extravirgin olive oil
 (EVOO) are due to the combination of high content of monounsaturated fatty acids (MUFA) and
 polyphenols. The EVOO Coratina (EVOOC), produced in the Puglia Region within the Bari
 Province, is characterized by high values of polyphenols ranging between 400 and 1000 mg/kg
 of EVOO.

 What is unknown. The potential effects of EVOOC in people on HD have not been formally
 explored both for surrogate and patient level endpoints.

 Why this study. The investigators want to evaluate the effects of EVOOC in non-diabetic
 hypertensive patients undergoing chronic HD on a series of surrogate endpoints.

 AIM OF THE PROPOSAL The investigators will investigate the effects of EVOOC with high content
 of polyphenols compared with refined olive oil (ROO) on lipid levels in HD patients.

 STUDY DESIGN

 Participants:

 The investigators will enroll non-diabetic hypertensive and hyperlipidemic patients
 undergoing chronic HD Hypertension will be defined as systolic blood pressure >130 mmHg and
 diastolic blood pressure >80 mmHg or having any antihypertensive agents.

 Hyperlipidemia will be defined as LDL cholesterol values more than 70 mg/dl or receiving
 statins at any dose.

 Eligible patients may be of age. (40-80 years old)

 The following patients will be excluded:

 1. Baseline disease other than diabetic kidney disease or hypertensive kidney disease

 2. Severe heart failure (NYHA class III or IV)

 3. Patients with low compliance

 4. Smoking, intake of antioxidant supplements (eg, carotenoids, vitamin C, vitamin E and
 glutathione), aspirin, or any other drug with established antioxidant properties,
 hyperlipidemia, obesity (body mass index >30 kg/m2), diabetes, celiac or other
 intestinal disease, life-threatening

 5. Life expectancy <6 months based on managing physicians assessment

 6. Inability to participate in the trial based on managing physician assessment

 Interventions:

 The experimental intervention will be EVOOC. Participants will ingest two tablespoons of
 olive oil during the day: one spoon containing 10g of EVOOC (>5mg of total biophenols/kg of
 EVOOC) at lunch and one at dinner. Total biophenols ingested per day will be >10mg.

 Comparator:

 The control will be refined olive oil (ROO). Participants will ingest two tablespoons of ROO
 during the day: one spoon containing 10g of ROO at lunch and one at dinner.

 Cointerventions:

 Patients may be receiving any drug for anemia, bone disease, nutrition and other including
 dietary advice towards a Mediterranean dietary pattern.

 Patients may be on either chronic HD or hemodiafiltration (HDF). All treatments will be high
 flux.

 CASE REPORT FORM This includes demographic findings and laboratory data at each time visit
 during the follow-up. Moreover, inclusion and exclusion criteria will be present at
 randomization time Daily food and individual quantities will be recorded on 'ad hoc' designed
 case report form. The intake of food will be analyzed and converted into nutrients using the
 useful software Medisystems (Anthropometric variable -weight) will be measured during the
 study.

 Physical activity will be evaluated by the Minnesota Leisure Time Physical Activity
 Questionnaire. No restriction of physical activity will be given.

 Methods:

 This study will be a centrally randomized trial with blinding of participants, investigators
 and outcome assessors, based on the intention to treat principle.

 Organizational aspects:

 The study will be conducted by the Schena Foundation in collaboration with the Renal Unit at
 Policlinic,University of Bari, and the MED&FOOD spin-off of the University of Bari.

 Follow-up duration will be 12 weeks. Clinical visit and laboratory control will be done every
 4 weeks for 3 consecutive months.

 Randomization:

 Randomization will be performed centrally at the Schena Foundation, with appropriate
 stratification.

 Sample size:

 Sample size is opportunistic. The investigators will recruit a total of 24 individuals of
 whom 12 will receive EVOOC and 12 will receive ROO, based on the pilot nature of this study.
 There are, therefore, no formal sample size calculations.

 FOLLOW- UP Dietary and clinical monitoring will be done by nephrologists, cardiologists and
 dieticians. Participants in both groups will receive dietary counseling. The counseling will
 be accompanied by written information with detailed description of typical ingested foods and
 the use of specific questionnaires: 1) the 14-items for the adherence to the Mediterranean
 diet; 2) the 137-items for the food frequency; 3) the Minnesota questionnaire for the
 physical activity; 4) the short-form-36 for the quality of life.

 CARDIOLOGY INVESTIGATIONS

 -  Intima-media thickness study will be done by the measurement of the carotid-wall
 intima-media thickness (cIMT). Echo-measurements will be made in three zones: (a)
 proximal zone: about 2 cm before the flow-divider; (b) distal zone: about ½ cm before
 the flow-divider; (c) middle zone. These data will be used to obtain the arithmetical
 mean cIMT value.

 -  Brachial artery reactivity study will be carried out by the ultrasonographic assessment
 of endothelial dependent flow-mediated vasodilation of the brachial artery. ECG

 -  24 hours ECG Holter monitoring

 -  2D Color Doppler Echocardiography Cardiological investigations will be held during the
 long intradialytic period except for the ECG Holter monitoring that will be done at
 least one hour before and four hours after dialysis.

 OUTCOMES

 Outcomes will be:

 -  cardiovascular event onset

 -  composite outcome: myocardial infarction, revascularization, ischemic stroke, peripheral
 artery disease, cardiovascular death

 -  blood concentration of LDL and HDL cholesterol, total cholesterol,

 -  blood pressure METHODS Blood samples will be drown in the morning after 12 hours of
 fasting. Urine sample will be collected from the first spot morning micturition in
 presence of residual diuresis.

 Blood and urine sample will be collected at the randomization visit and at each intervention
 period (every 4 weeks for a total of 3 months) after the short interval between two dialysis
 sessions.

 Peripheral blood mononuclear cells (PBMCs) will be collected for the genome study. Serum
 samples will be collected for the measurement of pro-inflammatory (IL-1b, IL-6, TNFa) and
 anti-inflammatory (IL4, IL-10) cytokines and CRP.

 A few ml of urine will be collected for tyrosol and hydroxytyrosol determinations by
 mass-spectrometry. In addition, other metabolites will be measured by nuclear magnetic
 resonance.

 Oxidative stress evaluation. The investigators will evaluate at T0 and after 12 weeks ROS
 serum concentrations by spectrophotometric method using commercially available kit (dROMs
 Test Diacron). The investigators will also assess the measurement of serum 8-oxodG, a marker
 of oxidative DNA damage by ELISA (Trevigen). The investigators will identify the nature of
 ROS generated in PBMCs by the peroxide-sensitive fluorescent probe
 2',7'-dichlorodihydrofluorescin diacetate (DCFH, Molecular Probes). Kinetic measurements of
 mitochondrial ROS generation will be evaluated by measuring the linear fluorescence increase
 caused by the H2O2-dependent oxidation of DCFH. NADPH-dependent superoxide generation will be
 measured by lucigenin-enhanced chemiluminescence methods in PBMCs isolated from HD patients.
 In parallel, the investigators will prepare RIPA lysates of the PBMCs and evaluate the
 expression of the various NADPH oxidase subunits by Western blot analysis. The antibodies for
 gp91phox/Nox2 or Rac1 antibodies will be furnished by Upstate/Millipore; p22phox, p47phox or
 p67phox will be purchased from Santa Cruz Biotechnologies. Additionally, the activity of the
 small GTPase Rac will be evaluated by affinity precipitation using the p21-activated kinase
 (PAK-1) binding domain linked to glutathione S-transferase (GST-PAK-1-PBD) on
 glutathione-agarose beads (Millipore/Upstate Biotech.).

 STATISTICAL ANALYSIS Normality of variable distribution will be assessed by analysis of
 skewness and kurtosis. One-factor ANOVA and Kruskall-Wallis test will be used to analyze the
 differences among the two groups (case-control).

 The general linear model for repeated measurements will be used with multiple paired
 comparisons in order to assess differences between the groups.

 Linearity of values across refined OO and EVOO will be determined for testing the
 dose-response effect of biophenolic compounds.

 Sample size and power calculations are not applicable given the pilot nature of the study
 with recruitment of an opportunistic sample.

 Primary statistical analysis: All analyses will be carried out on an intention-to-treat
 basis. Statistical significance will be defined as p<0.05 for a two-sided test.

 CHARACTERISTICS OF EVOOC AND ROO EVOOC: This olive oil is obtained by Coratina cultivar trees
 that are DNA certificated.

 ROO: This olive oil used as placebo is a mixture of refined OO. Fatty acid composition and
 polyphenol content have been determined by gas chromatography and HPLC, respectively.

 Polyphenols are undetectable in ROO. The characteristics of EVOOC and ROO (nutraceutic label)
 are enclosed in this protocol.

 The OOs will be provided by the MED&FOOD spin-off, University of Bari.
Inclusion Criteria:

 -  The investigators will enroll non-diabetic hypertensive and hyperlipidemic patients
 undergoing chronic HD

 -  Hypertension will be defined as having a systolic blood pressure of >130 mmHg and a
 diastolic blood pressure >80 mmHg or having any antihypertensive agents.

 -  Hyperlipidemia will be defined as having LDL cholesterol values more than 100 mg/dl or
 receiving statins at any dose.

 -  Eligible patients may be of any age.

 Exclusion Criteria:

 -  Diabetes mellitus type 1 or 2

 -  Severe heart failure (NYHA class IV)

 -  Patients with low compliance or severe psychiatric illnesses

 -  Smoking, intake of antioxidant supplements (eg, carotenoids, vitamin C, vitamin E and
 glutathione), aspirin, or any other drug with established antioxidant properties,
 hyperlipidemia, obesity (body mass index >30 kg/m2), diabetes, celiac or other
 intestinal disease, life-threatening

 -  Survival expectancy <6 months based on managing physicians assessment

 -  Inability to participate in the trial based on managing physicians assessment