Bioavailability of Oleanolic Acid Formulated as Functional Olive Oil
Condition: Healthy Subjects; Bioavailability
Study type: Interventional

Summary: 
      The oral bioavailability of Oleanolic acid (OA) when formulated as functional olive oil, and
      its mechanisms of systemic transport, will be approached by mean of randomized and controlled
      trial with 20 healthy volunteers. Ten individuals randomly selected will receive 55 mL of the
      functional OA-enriched olive oil (equivalent dose 30 mg OA) as part of an experimental
      breakfast. The other ten participants will receive within this experimental meal the same
      amount of the control olive oil. Immediately before and after eating the respective
      breakfasts, aliquots of cubital blood will be drawn every hour, over a postprandial period of
      7 hours. Since in this trial design, each participant is his/her own control, a four-week
      washout period is established, after which a new series of tests that cross the type of olive
      oil consumed will be carried out.

      From the aliquots of cubital blood, sera will be obtained by centrifugation. The extraction
      and quantification of serum OA will be realized by gas chromatography (GC) using flame
      ionization (FID) and mass spectrometry (MS) detectors. In the pharmacokinetic analysis of
      data, a mono-compartmental model will be assumed. It will be determined: 1) absorption
      parameters such as the maximum concentration achieved and the timing for it, the constant of
      absorption and the area under the curve; 2) distribution parameters such as the constant and
      volume of distribution; 3) metabolism parameters, such as the OA fraction associated with
      albumin; and 4) elimination parameters such as the elimination constant, the half-life and
      the clearance.

      To demonstrate the presence of OA in postprandial TRL, chylomicron and VLDL fractions will be
      prepared by plasma ultrafiltration in normal saline, and hydrolysed with pancreatic enzyme.
      The possible presence of OA among the TRL-derived lipids will be evaluated. The content of
      apo B48 and B100, as markers of the presence of chylomicrons and VLDL, respectively, will be
      determined by ELISA.

      Other parameters related to glycemic control, such as serum insulin, C-peptide and GLP-1 will
      be analyzed by ELISA.
    

Detailed description: 
      Design and implementation of the biovailability assays The study of oral bioavailability of
      Oleanolic acid (OA) when formulated as functional olive oil, and its mechanisms of systemic
      transport, will be approached by mean of a postprandial intervention with 20 healthy
      volunteers.

      The participants will be recruited after the complete biochemical and hematological analysis
      yield results within normal limits, and after checking in their electronic medical histories
      that they do not suffer from digestive, metabolic or oncologic disorders or any other
      pathology that, in opinion of the clinical investigators, could prevent them from
      participating in the trial. For their inclusion in the trial, it is also required that they
      grant the written consent to the protocols approved by the Institutional Committees for Human
      Research, after being conveniently informed both orally and documentally.

      The fieldwork will be performed in the Day Hospital of the Endocrinology and Nutrition
      Service of the University Hospitals 'Virgen del Rocio' from Seville (SPAIN).

      Sample size. The main quantitative variable to be examined in this trial is OA serum
      concentration. In the absence of consistent baseline data published so far, data from the
      analysis of OA in sera of the PREDIABOLE Study (ISRCTN03372660) have been used. A cohort of
      25 participants from the control group, consuming commercial olive oil, was selected, and an
      average baseline concentration of 155 ± 72 ng OA/mL was determined. Considering this, it is
      now postulated that the plasma OA content will increase by at least 50% as consequence of the
      intake of the functional olive oil (equivalent dose 30 mg OA). To prove this unilateral
      hypothesis test, with a confidence level of 90% (risk α=0.01, zα=1.645) and a power of 95%
      (zβ=1.645), a sample size of at least 17 volunteers is required. Assuming that losses lower
      than 15% are tolerable, the adjusted sample size would be 20 subjects.

      Anamnesis and physical examination of participants. The anamnesis will include vital data and
      general history collecting all the symptoms that the individual may present, their medical
      and surgical history, as well as pharmacological treatments followed. Information about
      lifestyle, perception of the own body image, diet and kind and intensity of physical activity
      will be also obtained. In addition, the muscle mass, fatty compartment (adipose panniculus),
      existence of edema, etc. will be examined. Likewise, systolic and diastolic blood pressure
      and heart rate will be determined.

      Anthropometric study and body composition. The anthropometric study will include the
      determination of total body weight, height, body mass index (BMI), as well as waist and hip
      circumferences. The study and evaluation of these variables will be carried out in accordance
      with the guidelines of the International Society for the Advancement of kinanthropometry
      (ISAK). The study of body composition will be carried out by electrical bioimpedance, using a
      TANITA® body composition analyzer model BC-418MA. Fat mass, lean mass, muscle mass, total
      water, bone mass, basal metabolism and visceral fat will be quantified with this technique,
      making use of prediction equations validated and adjusted by age and sex.

      Biochemical studies. Samples of fasting cubital blood will be drawn to determine the
      parameters of general, hepatic and renal biochemistry. In addition, a complete urine analysis
      will be performed, which include the habitual physical, chemical and microscopically
      measurements. These determinations will be carried out at the Laboratory of Analysis and
      Clinical Biochemistry of 'Virgen del Rocío' University Hospitals of Seville.

      Intervention The participants will be encouraged to avoid the consumption of alcoholic
      beverages and tobacco during the day preceding the execution of the assays. On trial days,
      and after 12 hours of overnight fasting, blood samples will be drawn from the cubital vein.
      Subsequently, ten individuals randomly selected will receive 55 mL of the functional
      OA-enriched olive oil (equivalent dose 30 mg OA) as part of an experimental breakfast, that
      includes coffee, skimmed milk and whole-grain toast. The other ten participants will receive
      within the experimental meal the same amount of the control olive oil. After eating the
      respective breakfasts, aliquots of cubital blood will be drawn every hour, over a
      postprandial period of 7 hours. During this time, free access to water intake will be
      allowed.

      A four-week washout period is established, after which a new series of tests that cross the
      type of olive oil consumed will be carried out.

      Pharmacokinetic study of OA From the aliquots of cubital blood, sera will be obtained by
      centrifugation, and NaN3, PMSF and aprotinin will be added as preservatives, to avoid their
      proteolytic degradation. Serum samples will be stored at -80 0C in the laboratories of the
      IG-CSIC until analysis. The extraction and fractionation of serum OA will be realized by
      liquid/liquid and solid phase extractions, whereas the identification and quantification of
      the triterpene will be completed by GC-FID and GC-MS. In the pharmacokinetic analysis of
      data, a mono-compartmental model will be assumed. From data of the serum contents of free OA
      throughout the postprandial period the following parameters will be determined: 1) absorption
      parameters such as the maximum concentration achieved and the timing for it, the constant of
      absorption and the area under the curve; 2) distribution parameters such as the constant and
      volume of distribution; 3) metabolism parameters, such as the OA fraction associated with
      albumin; and 4) elimination parameters such as the elimination constant, the half-life and
      the clearance. For calculations, the SigmaPLot pharmacokinetic analysis module (Systat
      Software Inc., San Jose, CA, USA) will be used.

      Study of the presence of OA in triglyceride rich lipoproteins (TRL). Chylomicrons will be
      first isolated from plasma in normal saline by centrifugation at 100,000 × g for 20 min. Very
      low density lipoproteins (VLDL) will be subsequently separated at 230,000 × g for 18 h. The
      chylomicron and VLDL fractions will be hydrolyzed with pancreatic enzyme. The released lipids
      will be extracted with chloroform:methanol (2:1), and fractionated by solid/liquid extraction
      with methanol:chloroform (1:1) and isopropanol:acetonitrile:water (2:1:1). The free fatty
      acids (FFA) composition will be analysed by GC-FID as the correspondent methyl esters. The
      possible presence of OA among the TRL-derived lipids will be evaluated after fractionation
      and GC-MS analysis. The content of apo B48 and B100, as markers of the presence of
      chylomicrons and VLDL, respectively, will be determined by ELISA.

      Other biochemical parameters serum insulin, C-peptide and GLP-1 will be analyzed by ELISA.
    

Intervention name(s): Oleanolic acid (CAS no. 598-02-1; PubChem CID 10494); Control commercial olive oil
Intervention type(s): Dietary Supplement; Dietary Supplement
Intervention description(s): Postprandial study of bioavailability of Oleanolic acid, formulated as functional olive oil, in healthy subjects; Postprandial study of Oleanolic acid bioavailability in healthy subjects
Other intervention name(s): Oleanolic acid-enriched functional olive oil
Allocation: Randomized
Intervention model: Crossover Assignment
Masking: Double (Participant, Care Provider)
Primary purpose: Basic Science
Keywords: Oleanolic acid; bioavailability; triglyceride-rich lipoproteins; postprandial dietary intervention; pharmacokinetic; randomized and controlled trial; crossover design