Metabolism And Health Effects Of Citrus Limonoids in Hypercholesterolemic Humans
Condition: Hypercholesterolemia
MeSH terms: Hypercholesterolemia
Study type: Interventional

Summary: 
      Limonoids are compounds found in citrus juices, seeds and peel. Some research shows that they
      reduce blood cholesterol and also reduce the incidence of several forms of cancer. Although
      these compounds are part of the natural human diet, the dose required to provide health
      benefits and the risks associated with the intake of high doses by humans have not been
      studied. This study will examine metabolism, safety, lipid-lowering and anti-inflammatory
      effects of limonoids. The dose of limonoids in this study is equivalent to that found in six
      8 oz glasses of orange juice per day. The investigators hypothesize that limonin glucoside
      supplementation will reduce total cholesterol, LDL cholesterol, ratio of LDL and HDL
      cholesterol, number of LDL particles, and serum markers of inflammation.
    

Detailed description: 
      This is a 127-day study with a cross-over design, divided into three metabolic periods (P1,
      P2, and P3). For the first 14 days (P1), all subjects will receive two placebo beverages and
      will be asked to consume one each with breakfast and dinner. Then in metabolic period 2 (P2),
      subjects will be randomly assigned to one of two groups (Group 1 and Group 2). For the next
      56 days, Group 1 will continue to consume two placebo beverages per day, while Group 2 will
      consume two beverages containing limonoids (250 mg/beverage). During the final 56 days (P3),
      the beverages provided to Group 1 will contain limonoids, and beverages provided to Group 2
      will be placebo. Both the placebo and limonoid beverages will be 12-oz buffered, sterile,
      orange flavored, artificially sweetened beverage, containing 60 mg vitamin C, 11 mg
      riboflavin and zero calories. Subjects are instructed that they must drink all the beverage
      provided each day. Beverages will contain a biomarker (riboflavin), and the biomarker is
      assayed in urine samples collected every 2 weeks at the beverage pickup appointments to
      monitor compliance. The subjects will be informed that they are being monitored for
      compliance. Limonin glucoside is obtained from citrus waste materials utilizing
      chromatographic methods that employ food grade approved media and solvents. The final pure
      limonin glucoside is fractionally crystallized from a concentrated water solution of a
      chromatographically purified extract. The crystalline material is separated from the mother
      liquor, dried and subjected to mass spectral and nuclear magnetic resonance assessment. The
      spectral data is compared to data acquired for a pure limonin glucoside standard to establish
      final purity which is better than 95%.

      Blood samples (approximately 70 ml, less than five tablespoons) will be drawn after a 12 hour
      overnight fast on study days 1, 15, 71 and 127 by venipuncture. These blood samples will be
      used to determine serum limonoids, complete blood cell count (CBC), and comprehensive
      chemistry panel (CCP), serum lipids, markers of inflammation, lymphocyte and monocytes
      functions. In addition to the fasting blood draws on days 15 (Group 2) and day 71 (Group 1),
      repeated blood samples (5 ml each time, approximately one teaspoon) will be drawn at 2, 4, 6,
      8, and 24 hr after taking two 12-oz limonoid beverages (total limonoids 500mg) to determine
      the absorption and metabolism of limonoids. The investigators note that the blood samples
      drawn 24-h after the limonoid intake will be on study day 16 for Group 2 and day 72 for Group
      1. The total amount of blood drawn will be less than 500 ml (2 cups) in 127 days. A spot
      urine sample will be collected at the same visit as the fasting blood draws, and every two
      weeks at the beverage pickup appointments. Repeated blood draws at 0, 2, 4, 6, and 8 hr after
      the limonoid supplement will be accompanied by the collection of urine samples. No food will
      be allowed during the 8 hrs; water intake will be monitored and restricted to one liter.
      Subjects will be served two standardized meals during this time, each representing 35% of
      their daily caloric intake, one after the 0 hr blood draw and the other after the 8 hr blood
      draw.

      Subjects will be instructed not to change their diets, exercise or lifestyle during the
      course of the study. They will also be instructed not to consume any citrus fruit/juice
      (orange, grape fruit) throughout the study. Three 24-hour dietary recalls will be collected
      by telephone during the last week of each metabolic period (P1, P2, P3). A standardized diet
      will be served for all three meals for each day prior to the blood draws.
    

Intervention name(s): Citrus Limonoid Beverage; Placebo beverage
Intervention type(s): Dietary Supplement; Dietary Supplement
Intervention description(s): The limonoid containing beverage will be 12-oz buffered, sterile, orange flavored, artificially sweetened, containing 60 mg vitamin C, 11 mg riboflavin and 250 mg limonin glucoside.; The placebo beverage will be 12-oz buffered, sterile, orange flavored, artificially sweetened, containing 60 mg vitamin C and 11 mg riboflavin.
Allocation: Randomized
Intervention model: Crossover Assignment
Masking: Double (Participant, Investigator)
Primary purpose: Basic Science
Keywords: hypercholesterolemia; inflammation; citrus limonoids