Effects of Lentinula Edodes Bars on Dyslipidemia and Oxidative Stress in Cholesterol Individuals: Randomized Study

2.1 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).

2.2 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 (BRASIL, 2006).

2.3 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 (KERCKHOFFS et al., 2003).

2.4 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 30 days - T30) and in the third (Time 60 days - T60) 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.

2.5 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 (GROTTO et al., 2015), 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 30 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 (T30). At the end of sixty days (T60), subjects returned for the last blood collection and final nutritional assessment.

2.6 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 (FRIEDEWALD et al., 1972).

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.

2.7 Oxidative Stress Analysis The determination of reduced glutathione (GSH) was made by quantification of reduced total thiols based, according to the Ellman method (1959). 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, the method of Aebi (1984) 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, according to Ohkawa et al. (1979). 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).