Effects of Lentinula Edodes Bars on Dyslipidemia and Oxidative Stress in Cholesterol Individuals: Randomized Study
Condition: Dyslipidemias
MeSH terms: Dyslipidemias
Study type: Interventional

Summary: 
      The daily change in population routine has stimulated the development of health beneficial
      value added products. Lentinula edodes (Shiitake) is a mushroom with high protein
      concentration, low lipid content, rich in fiber, minerals, vitamins, antioxidant compounds,
      β-glucans and chitosan. This is clinical study phase II, randomized, double-blind for analyze
      in the effect of eating Shiitake bars on cholesterolemia and oxidative stress levels in
      individuals with borderline cholesterol. Individuals with at least one of the following
      biochemical markers (total cholesterol, LDL or triglycerides) at the borderline level were
      recruited through online questionnaire. Individuals (n = 68) were randomly allocated to two
      groups (Group I - Shiitake free bar (n = 32); Group II - Shiitake bar (n = 36). Each
      individual underwent blood collection at 0, 33 and 66 days, and received an unidentified
      opaque bag containing the bars.Biochemical analyzes (triglycerides, total cholesterol, LDL,
      HDL and glucose) and oxidative stress markers (Catalase, GSH and TBARS) were performed on
      samples of individuals.
    

Detailed description: 
      Study design The primary outcome of the study was cholesterolemia, as assessed by total
      cholesterol levels, triglycerides or low-density lipoproteins (LDL) and high-density
      lipoproteins (HDL). The secondary outcomes included blood glucose, Body Mass Index (BMI) and
      oxidative stress biomarkers.

      A prospective Phase II, randomized, double blind and placebo-controlled trial was conducted
      from September 2018 to December 2018. The study accomplished at University of Sorocaba
      (UNISO), São Paulo State, Brazil. The study was approved (Annex D) by Human Research Ethics
      Committee from University of Sorocaba (protocol number 2.824.297), in accordance with
      Resolution 466/2012 of the National Health Council (BRASIL, 2012) and Research Ethics Review
      Committee (ERC) (WHO, 2016).

      Recruitment of individuals For the recruitment of individuals, the disclosure was initiated
      five months before the start of Phase II trial. The research was published on the website of
      the University of Sorocaba (UNISO), on social media and folders distributed throughout campus
      containing a brief explanation about the research, inclusion and exclusion criteria, and
      contact of the researcher. According to the Resolution 1.170, from Anvisa (National Health
      Surveillance Agency), Phase II studies are not allowed with less than 12 people; however, the
      Agency suggests using a minimum of 24 volunteers.

      Eligibility Criteria A total of 165 individuals completed the Google online survey.

        -  Inclusion Criteria Individuals aged from 20 to 65 years old, of both gender, who had at
           least one of the following biochemical markers at the borderline level (total
           cholesterol 180 to 239 mg/dL; LDL 130 to 159 mg/dL; triglycerides of 150 to 200 mg/dL),
           were recruited, diagnosed by biochemical exams with dates recent to the day of
           recruitment. The be tolerant to bars ingredients and to Shiitake. Availability to attend
           the date and time of blood collection.

        -  Exclusion Criteria However, some of them had diseases such as cancer, heart disease,
           neurodegenerative disease, diabetes, among others. These diseases could be considered as
           confounders of the study and some recruited people had to be excluded. Pregnant,
           lactating or hormone replacement women could not participate either.

      Individuals were instructed to no change their eating habits and patterns, physical activity
      level or oral contraceptive use during the study, thus identifying only the effects of
      Shiitake added to the diet for each individual on the exposed group.

      Study Interventions. The study officially started with 68 individuals, who signed an informed
      term of consent. In the first (Time 0 - T0), in the second (Time 33 days - T33) and in the
      third (Time 66 days - T66) meetings, the subjects were submitted to nutritional assessment
      and anamnesis, 24-hour recall of eating habits and eating frequency.

      In the first month of the study 4,100 placebo or mushroom bars were produced and in the
      second month the production was 3,500 bars. According to the Grotto and collaborators (2015),
      conducted a study evaluating different Shiitake concentrations (100, 400 and 800 mg/Kg/day)
      to determine the degree of toxicity in animals. Shiitake concentration of 100 mg/Kg/day has
      been shown to be safe. Besides, the Shiitake sweet bar formulation was approved in the
      sensory analysis and purchase intention in the previous study.

      Randomization The individuals (68) were randomly allocated (using a table of random numbers)
      into 2 groups: I - Placebo group (n = 32) and II - Intervention group (n = 36). To ensure the
      daily recommended intake of Shiitake of 100 mg/Kg/day, each participant was weighted to
      determine the number of bars consumed per day.

      The Individuals after nutritional assessment and blood collection, received an unidentified
      opaque and sealed packed containing individually vacuum bars for a period of T33 days. The
      patients and the data collector were blind. The bars of the intervention and placebo groups
      were similar in texture, flavor, aroma and appearance.

      After one month, all individuals returned to the University of Sorocaba for new nutritional
      assessments, receiving more food bars and blood collection with the same double-blind care
      (T33). At the end of sixty-six days (T66), subjects returned for the last blood collection
      and final nutritional assessment.

      Biochemical Analysis Total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides and
      glucose were analyzed by commercial kits, following the manufacturer's specifications, in the
      automated equipment COBAS C111 (Roche®) according to the methodologies below.

      For cholesterol, 10 µL of sample and 1.0 mL of enzyme reagent were mixed at 37 ºC for 10 min
      for total transformation into free cholesterol, by lipoprotein lipase enzyme. The reaction
      involved the oxidation of free cholesterol to cholesterol-3-one and H2O2 by the cholesterol
      oxidase enzyme. And with the action of peroxidase on phenol with 4-aminoantipyrine formed a
      cherry chromogen, the color being proportional to the concentration of cholesterol.

      For the determination of HDL, the serum was treated with phosphotungstate acid and magnesium
      chloride. So, the LDL was precipitated out from the fraction after centrifugation for 2 min
      at 10.000 rpm. HDL, which remained in the supernatant, was performed according to the
      cholesterol methodology. HDL concentration is proportional to the color. LDL was calculated
      by subtracting HDL cholesterol from total cholesterol.

      Serum triglycerides were hydrolyzed to glycerol and free fatty acid by lipoprotein lipase. In
      the presence of adenosine triphosphate (ATP) and glycerol kinase, glycerol-3-phosphate was
      phosphorylated, which was oxidized to acetone dihydrogen phosphate and H2O2 by the glycerol
      phosphate oxidase. H2O2, 4-aminoantipyrine and p-chlorophenol underwent the same reaction
      process reported above by peroxidase in a 37 ºC water bath for 10 minutes until formation of
      red color.

      For the blood glucose test, glucose-6-phosphate dehydrogenase oxidized glucose-6-phosphate in
      the presence of NADP in gluconate-6-phosphate. No other carbohydrates have been oxidized. The
      rate of NADPH formation during the reaction was directly proportional to the glucose
      concentration, being determined photometrically.

      Oxidative Stress Analysis The determination of reduced glutathione (GSH) was made by
      quantification of reduced total thiols based. For this, 150 µL of the blood was vortexed with
      100 µL 10% Triton X-100 and 100 µL 30% TCA, then centrifuged for 10 min at 4000 rpm. In the
      cuvette 900 µL of 1M TFK, 50 µL of supernatant, and 50 µL of 10 mM DTNB were pipetted, which
      reacted to form a yellow complex. The reading was taken at 412 nm in a spectrophotometer. For
      the calculation of concentration, the calibration curve with predefined GSH concentrations
      (0.005; 0.01; 0.025; 0.05; 0.1 mM) was used.

      To evaluate the activity of the catalase enzyme was used, based on the decomposition of H2O2
      by catalase, monitored at 240 nm. For this, the blood was diluted 60 times in 50 mM TFK. An
      aliquot of 20 μL was mixed to 1910 μL of the same TFK, and 70 μL of H2O2 was added,
      initiating the monitored reaction for three minutes. A constant of variation (κ) per minute
      helped in the expression of catalase enzyme (κ/min).

      Thiobarbituric acid reactive substances (TBARS) were used as a biomarker of lipid
      peroxidation. Plasma aliquots (150 µL) were mixed with 50 µL NaOH and 50 µL Milli-Q ultrapure
      water (Direct 8, Millipore®) and incubated at 60 °C for 30 minutes with shaking. 250 µL of 6%
      H₃PO₄, 250 µL of 0.8% TBA and 100 µL of 10% SDS were added to the samples and taken to the 80
      °C bath for one hour. Lipid peroxides reacted with TBA in acidic medium to form a pink
      compound and read in a spectrophotometer at 532 nm. To calculate the concentration of TBARS
      in plasma, a calibration curve of malondialdehyde, the main thiobarbituric acid reactive
      substance, was made (0.28; 0.56; 1.7; 3.4; 6.6 µM).
    

Intervention name(s): Shiitake cereal bar
Intervention type(s): Other
Intervention description(s): patients with borderline cholesterol exposed to bars with Shiitake
Allocation: Randomized
Intervention model: Parallel Assignment
Intervention model description: Prospective, randomized, double blind and placebo-controlled
Masking: Triple (Participant, Investigator, Outcomes Assessor)
Primary purpose: Treatment
Keywords: Lentinula edodes; Clinical study; Cholesterolemia; Nutraceuticals