Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT02664428 |
| Other study ID # |
PreBIOil 2 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
N/A
|
| First received |
January 16, 2016 |
| Last updated |
January 11, 2017 |
| Start date |
November 2015 |
| Est. completion date |
January 2017 |
Study information
| Verified date |
January 2017 |
| Source |
Fondazione Edmund Mach |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
Italy: Ethics Committee |
| Study type |
Interventional
|
Clinical Trial Summary
Study hypothesis: Diet integrated with food prepared with olive, buckwheat, peas and
chestnut flour as in PreBIOil product combination can modify the gut microbiota and the
cholesterol metabolism.
Primary objectives of the study are to assess whether the test product to be able to change:
1. fecal microbiota profile;
2. Plasma cholesterol LDL, total, total LDL and HDL ratio;
3. plasma triglycerides;
4. Apolipoprotein ApoA-I, ApoB, and Lp.
Secondary objectives of the study are:
1. anthropometric indices;
2. secondary metabolites of polyphenols in human biofluids;
3. mass spectrometry plasma and urine metabolite profile;
4. blood glucose and fasting insulin levels; 5,6) C-reactive protein (PRC or hsPRC);
7) urinary isoprostane F2; 8) oxidized LDL in plasma.
Study Design: placebo-controlled, randomized, double-blind parallel trial.
Inclusion criteria: Aged 30-65 years; BMI 20-29,9 kg/m^2. Total Cholesterol 180-240 mg/dl
Exclusion criteria: Fasting blood glucose >150 mg/dl; triglycerides >500 mg/dl; uncontrolled
hypertension (blood pressure [BP] >160/100 mm Hg under antihypertensive therapy); any long
term medical therapy; food intolerances; alcohol intake >5 drinks per day or use of narcotic
substances; use of dietary supplements, pro or pre- biotics; special diet; pregnancy,
tobacco smoking.
Methodology: Determination of eligibility: for each volunteer aged between 30 and 65 years,
will undergo to clinical and biochemistry evaluation. Clinical visit, clinical tests, and
blood drawing will be performed after an overnight fasting at visit T-1 at the Casa di Cura
Eremo di Arco (TN). In this T-1 visit the eligibility will be established and the
participants will be randomized to receive supplementation with either PreBIOil biscuit
(90g/day) or Control for 8 weeks in a double-blind manner. Clinical tests, blood drawing,
and stool and urine collection will be performed during visits at the beginning and end of
each treatment period (T0 and T1). A 4 day-food diary record will be collected before visits
T0, at the beginning of T0 and before T1.
Efficacy Assessments Arterial BP; BMI; ratio of waist to hip circumference; food
questionnaires; blood sample analysis (total cholesterol, triglycerides, HDL and LDL
cholesterol, oxidized LDL, serum glucose and insulin, C-RP, Apolipoproteins, Isoprostane;
urinary and plasma metabolite profiling; fecal microbiota analysis.
Safety assessments. Adverse events registration.
Statistical analyses. The differences between the two group will be evaluated with
univariate and multivariate statistical methods. The medium before and after the
intervention will be compared with the General Linear Model (ANOVA) for repeated measures or
for paired data. Simple and multiple linear regressions will be performed to determine the
relationships between independent variables; also we will run the t-test to evaluate
differences in compliance detected in the types of supplementation. The results are
expressed as mean +/- SEM and the differences will be considered significant when P <0.05.
Duration: Subjects will make three visits (visit T-1, beginning visit T0, and end of
treatment period visit T1, week 8 from T0). The treatment duration is 8 weeks; a daily
portion of about 90 g of biscuit is introduced.
Description:
The first results of in vitro testing of the project PreBIOil have delineated the capacity
of a flour of olives to modulate the microbial population of the human intestine in a manner
comparable to that of inulin recognized as polysaccharide with a capacity prebiotic. Based
on these results, and to others in the literature, the bakery product PreBIOil has been
formulated. The product contains, flour of olives, buckwheat, pea, and chestnut.
The overall objective of the study is to assess whether the introduction in the diet of a
bakery product prepared with olive flour, mixed with buckwheat, pea and chestnuts flour can
change the profile of the intestinal microbiota and the plasma cholesterol, in particular
LDL. The study is set up as dietary supplementation, double-blind, randomized, parallel with
the control group, in which the following product will be compared:
i) a new bakery product designed study PreBIOil (test); ii) a bakery product control
isoenergetic (control).
Description of the Food supplemented in the Trial The test product is new baked product
formulated with a mixture of flour from olives, buckwheat, pea and chestnut and has the
appearance of a salty biscuit.
Participants will be required to consume daily about 90 grams of test or control product.
The exact quantities of each product batch will be determined following the assessment of
the nutritional characteristics of the finished product in order to provide the same calorie
intake with the two products.
Digestibility, fermentability and modulation of the microbiota Buckwheat is considered a
non-cereal that in addition to containing fiber, minerals and vitamins, such as B2 and B6,
also represents a source of antioxidant compounds due to the content of flavonoids.
The pea flour contains important components for human diet (starch, protein and other
nutrients). The health benefits associated with consumption of peas primarily derived from
the concentration and properties of starch, protein, fiber, vitamins, minerals and
phytochemicals. The fiber contained in the integument and in the cell walls of the cotyledon
contributes to gastrointestinal function. The starch content in pea flour is characterized
by lower glycemic index. The proteins from pea may produce peptides with antioxidant
activity. The vitamin and mineral content of peas can play an important role in the
prevention of deficiency diseases, in particular those relating to the deficiencies of
selenium and folate. Peas contain a variety of phytochemicals. These include polyphenols
that may have antioxidant and anticancer, saponins which are present hypo colesterolemica
and anticancer activities, and oligosaccharides of galactose that can exert prebiotic
benefits in the large intestine.
The nutritional components of chestnut flour consist of carbohydrates, protein and fat in
the composition such as to provide an energy value about 350-400 kcal / 100 g. Chestnuts
have been voted to have a positive effect on the metabolism of gut bacteria and are a source
of long-chain fatty acids C18: 1 and C18: 2 and vitamins (vitamin E). The possible presence
of polyphenols is limited to the outer skins, generally separated from the fruit during the
production of the flour. The flour coming from olive paste is not described in the
scientific literature for specific nutritional properties for the human diet. However olives
are an integral part of the Mediterranean diet in which they are consumed as table olives
and olive oil. The olive fruit contains 50% water, 22% fat, about 19% of carbohydrates and
about 2% of protein; the polyphenol content varies in the range 1-3%. The olives are
consumed or as olive oil or table olives. The olives are characterized by strong bitterness,
caused mostly dall'oleuropein. The oleuropein can reach 140 mg / g dry weight in unripe
olives and decreases and as the fruit ripens Table olives are treated to mitigate the bitter
taste through processes such as maceration soda and fermentation in brine. However in the
process of sweetening the bitterness over lost some important compounds present in olives
such as polyphenols and fiber. There is no evidence on the ability of extra virgin olive oil
polyphenols reducing the risk of cardiovascular disease, but a large part of the possible
effects of the components of the olive alone or in synergy with the polyphenols are still
unexplored. In addition a large part of the water-soluble fraction contained in the olives,
between which the fibers and polyphenols with proven beneficial activities on human health,
is not removed in the process of oil production. There is evidence that some phenolic
fractions as secoiridoids are poorly absorbed by the intestinal mucosa and are then
available for the colonic microflora. Essentially the oil in its entirety would have the
greatest potential as a food can modulate beneficially the composition and activity of
microorganisms symbiotic human gut, as observed experimentally for other types of fruit.
By means of simulation in vitro of colonic fermentation it has been observed that the olive
paste, even after baking at 180°C for 30 ', is capable of modulating the human intestinal
microbiota with a prebiotic effect or inducing a significant increase of bifidobacteria.
Such simulation in vitro was carried out by incubation for 24 h in a batch fermentation
system with control of pH and temperature, following inoculation with feces obtained from
five healthy individuals. The analysis of total polyphenols (main antioxidants present in
the test product) present in the flour of olives, in the mixture of the flour and in the
test product did detect a loss of total polyphenols, as a result of baking equal to 40%.
Clinical Results Clinical results have shown that daily consumption of 100 g of buckwheat in
the form of flour has determined the increase in the HDL/cholesterol ratio and improved
glucose tolerance and more recently it was observed that the consumption of biscuits made of
buckwheat has reduced the serum level of a marker of inflammation, myeloperoxidase (MPO) in
addition to a reduction of total cholesterol with better pulmonary vital capacity. A recent
meta-analysis of several intervention studies on the human diet with supplementation of
legumes, including peas, showed their ability to decrease serum levels of LDL cholesterol.
Intervention studies on diet with olive oils, by-products of processing of olives and
olive-based products have been conducted with healthy volunteers and subjects with various
degrees of cardiovascular risk. Of these studies have been indicated the daily doses of
total polyphenols introduced (from a few mg / day to 30 mg / day, up to 100 mg / day)
depending on the type of olive oil and also enrichment with extracts. These interventions
had in many cases a duration of about a month, and most of the studies measured the effects
of antioxidant and anti-inflammatory changes in the circulatory system, along with the
values of plasma lipids, and other parameters related to oxidative stress. In particular in
the study "EUROLIVE", on a large sample of healthy individuals, it was observed that the
phenols of olive oil were able to protect blood lipids from oxidation, increase the
concentration of HDL cholesterol and the relationship between reduced and oxidized
glutathione, however no changes dell'F2-isoprostane.
In 2011 the European Food Safety Authority (EFSA) has approved the claim that the olive oil
polyphenols, especially hydroxytyrosol, protect LDL from oxidation, at a dose greater than
or equal to 5 mg/kg/day. Other studies have also shown that olive oil polyphenols are
related to the reduction of CRP, reduced atherogenic gene expression in blood cells, and
decreased blood pressure. Although there are evidences on the ability of olive oil
polyphenols to reduce the risk of cardiovascular diseases, a large part of the possible
effects of the components of the olive alone or in synergy are still unexplored. Moreover, a
large part of fibers and polyphenols contained in the olive is not extracted in the process
of oil extraction and eliminated as a byproduct.
Rationale of the Study The buckwheat flour adds fiber and flavonoids, the pea flour contains
carbohydrates with low glycemic index, protein and is also a source of antioxidants,
chestnut flour brings prebiotic substances and vitamins. Flour olive is the fruit of the
olive is with its water-soluble components (eg. Fibers and polyphenols) and in the soluble
fraction of polyunsaturated and monounsaturated fatty acids, tocopherols and carotenoids
that only partially pass extra virgin olive. These components are designed mostly as
components of extra virgin olive oil and scientifically have given evidence to contribute to
the improvement of lipid metabolism and plasma lipids framework, to heart health and protect
against oxidative stress.
Based on available clinical and experimental studies, it has been suggested that consumption
of a baked product made from a mixture of flour from olive paste, buckwheat, peas and
chestnuts can have positive effects on lipid plasma biomarkers and intestinal microbiota
(for example, an increase of bifidobacteria) in adult participants with plasma content of
cholesterol in medium to high levels even if not yet pathological.
Statement of compliance statement This study will be carried out in compliance with the
Protocol and in accordance with Good Clinical Practice (GCP), as described in ICH GCP for
1996, the US Code of Federal Regulations (CFR) and the Declaration of Helsinki. With the
signing of this protocol, the researcher agrees to comply with these requirements.
Objectives of the study
The overall objective of the study is to assess whether the introduction in the diet of a
new bakery product containing a mixture of flour from olive paste, buckwheat, peas,
chestnuts is able to change:
Primary objectives:
1. The profile of fecal microbiota;
2. Plasma levels of total cholesterol, LDL, ratio / total LDL and HDL;
3. The plasma levels of triglycerides;
4. Apolipoprotein ApoA-I, ApoB, and Lp (a).
Secondary objectives:
1.The anthropometric indices 2 The secondary metabolites of polyphenols in human biofluids
3.The profiles of metabolites in plasma and urine assessed by analysis of mass spectrometry
(MS); 4. The blood glucose and fasting insulin levels; 5. C-reactive protein (PRC); 6.
High-sensitivity C-reactive protein (hsPRC); 7. urinary isoprostane F2; 8. oxidized LDL in
plasma.
Presentation experimental design Participants volunteers will be identified and recruited at
an outpatient part of the Casa di Cura Eremo di Arco (TN). Participants will be informed
about the purposes and methods of the study and will be pre-selected based on criteria for
inclusion / exclusion. The participant, will sign the informed consent and will enter the
studio after the visit to T-1, performed at outpatient part of Casa di Cura Eremo di Arco
(TN) where it will be performed a clinical and biochemical health status. To allow proper
monitoring of eating habits, the eligible participant, after the first visit will fill a
food diary.
If subjects will be eligible for the study based on all the criteria for inclusion and
exclusion, will be randomized to receive either placebo or the active integration at visit
T0.
The clinical examination, and the collection of blood and urine will be carried out after
fasting for about 10 hours at the beginning of the start-up period (visit T-1). Clinical
tests, blood samples, collection of feces and urine will be performed during visits T0, T1.
Additional food diary (4 consecutive days) will be filled at visits T0 and ahead of T1.
Adverse events, taking medications and supplements will be recorded by the participants in a
special diary.
Participants included in the study will also be asked to fill in a sensory evaluation of
each food offered in blind, the first taste and at the end of 8 weeks of supplementation for
each product
Study Procedure Recruitment of participants Participants will be identified and recruited at
an outpatient part of Casa di cura Eremo di Arco (TN) Participants will be informed about
the purposes and methods of the study and will be pre-selected based on criteria for
inclusion/exclusion. If eligible, the participant will sign the informed consent and will
enter the studio with a visit to T-1, which will be performed during a clinical and
biochemical state of health. The subjects that will be eligible for the study based on all
the criteria for inclusion/exclusion, will be included in the study and randomized to
receive the first food supplementation (test or control) to visit T0 (two weeks later by the
run-in).
Supplementation
After eligibility is assessed, the participants will be randomized to receive the
integration with the bakery product with the test or control product:
The single daily doses will be packaged in packs encoded by a qualified group.
Supplementation will last eight weeks, double-blind, parallel. with one of the two products
(attributed to randomization).
Follow up The project will consist of a period of eight weeks of supplementation, clinical
examinations and the collection of blood and urine will be made after an overnight fast
(excluding water consumption) of about 10 hours to visit T-1 . Clinical tests, blood
collection, the collection of feces and urine tests will be carried out visits to T0, T1: T0
is the day of the first integration, T1 will be eight weeks after the start of treatment.
The food diary will be compiled to visit T-1 and distributed before visits T0 and T1 for
data collection visits T0 and T1.
Clinical evaluation Electrocardiogram. An ECG will be performed at screening (T-1). The ECG
tracings will be reviewed and interpreted by the outpatient part of the nursing home
Hermitage of Arco.
Blood pressure will be measured according to the guidelines for hypertension ISH / WHO 2004
Anthropometric indices Weight and height will be measured on a standard scale. The measures
will be carried out on subjects without shoes, coats or warm clothes. Body-mass index, BMI,
is calculated as kg /m2.
The waist circumference (umbilical) will be measured in a manner determined by the National
Institutes of Health, National Heart, Lung, and Blood Institute.The ratio of the
circumference of the waist and hip ratio (WHR) is calculated.
Questionnaires All information will be collected and recorded in validated questionnaires,
including personal identification, medical history and information on the risk factors for
cardiovascular disease, medication use, physical activity and eating habits. Also a four-day
food diary will be written by each participant.
Participants included in the study will also be asked to fill in a sensory evaluation of
each food offered in the first blind tasting and at the end of 8 weeks of supplementation.
Sampling of blood and urine collection The blood (≤ 30 ml) will be drawn from a peripheral
vein, after 10 hours of fasting. 24 h urine will be collected, the volume measured and
sample will be stored at -80 ° C.
Collection of fecal samples Fecal samples will be processed within 12 hours of sample
collection. Aliquots of each fecal sample will be frozen directly at -80 ° C for analysis of
metabolomic profile.
Laboratory tests by the laboratory analysis of the Casa di Cura Eremo di Arco Blood samples
will be centrifuged according to the requirements of the tests to be performed immediately
after blood collection. Plasma or serum will be separated, in part immediately tested and
the remaining fraction stored at -80 ° C.
Total cholesterol, HDL cholesterol, triglycerides and glucose will be evaluated with
enzymatic method. LDL cholesterol will be measured analytically with enzymatic method.
Insulin levels will be assessed by elettrochemioluminescenza. The blood count and platelet
count will be assessed by method impedentiometric. The C-reactive protein (CRP) will be
measured by turbidimetric assay. The high-sensitivity C-reactive protein (hsPCR) will be
measured by turbidimetric assay.
Apolipoprotein AI (ApoA-I), ApoB, and Lp (a) were determined by immunoturbidimetry.
The test for the verification of the absence of glucosuria will be made by measuring the
concentration of glucose in the urine.
Laboratory tests by the Fondazione Edmund Mach [EMF] Urinary and plasma metabolomic profile.
The F2-isoprostanes will be measured in the urine as a measure of oxidative stress, by means
of specific EIA kit, after purification by chromatography solid phase (columns SepPack).
The analysis of LDL oxidized in the plasma will be conducted with the aid of ELISA kit.
The samples of urine and blood (plasma and serum) will be subjected both to analysis of
targeted metabolomics (UPLC-MS / MS for non-volatile compounds and GC-MS / MS for volatile
compounds) that analyzes of non-targeted metabolomics (ULPC-Q -TOF-MS and Nano -FTMS-Q-TOF
nonvolatile and GC-TOF for the volatile compounds). For profiles of targeted plasma
metabolites, it will be used the MS-based Biocrates AbsoluteIDQ ® Kit P180, generating
precise quantitative data on 180 human metabolites linked to the risk of diabetes. In
addition, specific targeted analysis will be applied to the lipid profile of serum bile
acids, short-chain fatty acids (short chain fatty acids, SCFAs) and quantification of
metabolites of the polyphenols of microbial origin in biofluids (including feces) with UPLC
/ QQQ-MS / MS.
Analysis of fecal microbiota. Changes in the faecal microbiota of specific bacterial
populations will be evaluated using the method of in situ hybridization with fluorescent
probes (FISH).
Also changes in the faecal microbiota will be evaluated using a metagenomics approach using
pyrosequencing 454. In short, after the collection of stool samples (n = 128, 2 stool
samples at different times T0, T1 from 64 subjects before and after consumption of each food
test and placebo) will extract the bacterial genomic DNA using the kit SPIN FastDNA (MPbio)
specific for feces. Using primers specific for the bacterial 16S rRNA gene (hypervariable
region V1-V3) it will be performed by PCR amplification and then subjecting the amplified
V1-V3 at 454 pyrosequencing.
Will use the GS FLX Titanium Chemistry with adapters Multiplex Identifier (MID) to get
sequences of length 600 bp that will be analyzed by comparing the database of 16S rRNA
phylogenetic, publicly available, via the pipeline QIIME that allows assignment to the genus
level of human intestinal microbiota. It will be produced a total of about 20,000 sequences
for each fecal sample. As quality control and analysis of microbial ecology, it will be used
a multivariate statistical in order to correlate the changes in the composition of the fecal
microbiota following the consumption of food testing or placebo.
Randomization blinded Participants will be assigned randomly but equally distributed by sex
and age to one of two treatments: bakery product test or control product even using specific
software. The clinical researchers, investigators and laboratory personnel will be blinded
to the randomization.
Treatment The supplementation will be introduced in the daily diet through the consumption
of a bakery product (test) for a daily consumption of 90 grams of product containing flour
of olives, from buckwheat, from peas, chestnuts. The food will provide an analogue control
calorie intake. The products under study (test or control) will be supplied in packs that
will be labeled in the local language with the code number of the study. the manufacturer's
name, the number of patients, the number of study visit. The products will be distributed to
the participants from the visit T0.
Duration of study and planning After eligibility will be assessed, participants will be
randomized in two groups (product test or control) that will follow 8 weeks of dietary
supplementation.
The overall duration of the study, including the start-up period (run-in) and the treatment
will be 10 weeks.
The recruitment will start later and within three months of approval by the Ethics
Committee.
The run-in will begin within two weeks of the first visit (T-1). Subjects will be recruited
over a period of 10 months and the study will be completed within 12 months from the time of
the first recruitment.
Criteria for study discontinuation In accordance with the Declaration of Helsinki, ICH
standards of good clinical practice, and the "Food and Drug Administration (FDA)
Regulations" of the United States, a person has the right to withdraw from the study at any
time and for any reason, without prejudice for future medical care from the physician or the
institution. Investigators also have the right to withdraw the study subjects (see below).
In the event that a person (or a person legally authorized representative) decides to
retire, every effort will be made to complete and report the observations as well as
possible. A final comprehensive assessment should be made at the time of the withdrawal of
the subject where the reason for the withdrawal must be registered and in any case should an
attempt be made to perform a follow-up evaluation.
The Subjects can be removed from the study for the following reasons:
Breach of the Protocol as from the experimenter Not meaningful compliance by the subject The
refusal of the subject to continue treatment or comments Unacceptable toxicity Decision of
the researcher that the closure is in the best medical interest of the subject Unclear event
of illness or complications Use of concomitant medications that are incompatible with the
study. Pregnancy Abuse of alcohol or drugs Those who discontinue or is withdrawn from the
study will not be replaced.
Procedure for Terminating
If an individual stop before the completion of the study, the reason and the cutoff date
will be recorded. The date of the last dose of food in the study should be recorded. In the
event of early termination, the following procedures must be performed:
Clinical tests (systolic and diastolic, heart rate, weight and height to calculate BMI and
waist and hip circumference).
Taking blood and feces and urine collection. A food frequency questionnaire (weekly). In the
event that the individual can not return for follow-up visit, the investigators will have
groped to contact the person by phone to track any adverse events.
If a person withdraws consent to receive food in the studio, you will be encouraged to
remain in the study without receiving treatment for the collection of safety data.
The procedures of reliability of the product under study The products (test or control) will
be provided in packages labeled in the local language with the code number of the study, the
number of site code, the manufacturer's name, the number of participants, the number of
visits of the study, the number of lot, instructions for storage and expiration dates. The
products will be distributed to the participants from the visit T0.
Codes randomization The codes of randomization by sex and age, for each participant will be
stored in a sealed envelope by qualified group of Sensory Quality (QS of the Department of
Nutrition and Food Quality of the Research and Innovation Centre of the Edmund Mach
Foundation).
Data Source Control All data collected during the study will be obtained from primary
sources, and will be recorded in written documents and electronic medical records as the
subject. The results of clinical, laboratory results and ECG will be recorded in the
electronic medical record file of the subject. No data will be inserted directly in the
"case report form" (CRF), without being transferred from the primary source, such as paper
records or electronic.
Concomitant Drug Therapy Any medication taken from the subject within one month before the
start of the study and / or administered during the study will be considered as concomitant
therapy. Such treatment must be documented on the appropriate page of the CRF and the
medical records of the subjects. Any changes to the concomitant therapy during the study
must be clearly recorded and the reason for the change should be documented.
A letter will be sent to the doctor of each participant, specifying what are the treatments
concomitant incompatible with the study. The use of the following drugs will not be allowed
during the study: blood thinners, antihypertensives, lipid-lowering drugs, beta-blockers,
systemic corticosteroids, neuroleptics, antibiotics, laxatives, vitamin supplements,
antioxidants and minerals, dietary fiber, prebiotics (eg inulin, oligofructose) and
probiotics. Therefore if the subject requires drug therapies incompatible with the study,
the subject is notified by the treating physician to terminate the study and inform the head
of the study and following the procedures for the early termination.
Compliance Rating To assess compliance to supplementation, they will be measured urinary
phenols in subjects that will be supplemented with food tests at T0, T1. In addition, all
the unused or empty packaging at the end of each week will be returned to the experimenters.
Methods Security There were no security problems during previous studies related to the
consumption of products made from olives, buckwheat, pea flour, chestnut flour.
The study will be monitored by medical and paramedical staff of the Casa di Cura Eremo of
Arco. Adverse events will still be recorded throughout the study. Abnormal laboratory values
that induce clinical signs or symptoms or require therapeutic intervention will be promptly
notified to the subject, if the subject has explicitly expressed that wish to receive such
communication in the form of informed consent. The subject will be free to withdraw from the
study in advance if in need of therapeutic intervention is not compatible with the study, or
if you experience adverse events that cause a change in the criteria for inclusion of this
subject in the study. The subject will still be free to withdraw from the study at any time,
in case the study is due to discomfort following the procedure for early termination.
Statistical analysis The basic features are listed by type of supplementation: continuous
variables are expressed as mean +/- standard deviation or medians with interquartile ranges
and as a percentage for categorical variables. Data with an asymmetric distribution will
follow a log processing prior to analysis. The differences between the two supplements will
be evaluated with univariate and multivariate statistical methods. The medium before and
after the intervention will be compared with the General Linear Model (ANOVA) for repeated
measures or for paired data. You will perform simple and multiple linear regressions to
determine the relationships between independent variables; also we will run the test to
evaluate differences in compliance detected in the types of supplementation. The results are
expressed as mean +/- SEM and the differences will be considered significant when P <0.05.
Data with an asymmetric distribution will follow a log processing prior to analysis.
The main analysis will be based on a comparison of the differences in mean levels after
consumption of test product and after consumption of control. The differences between the
mean values at the end of each supplementation (T1) in relation to the relative mean
baseline value (T0) will also be measured.
The differences in time between treatment arms will be evaluated by analysis of variance for
repeated measures.The level of significance will be considered <0.05 in two tails.
The sample size for each supplementation has been determined to 32 to detect a decrease of
LDL cholesterol in the plasma equal to about 15.44 mg/dl with a standard deviation of 14.28
mg/dl using the equation Snedecor and Cochran with α equal to 0.05 and 1-β equal to 0.9
(Dell et al., 2002). According to this formula the minimum number of subjects to enroll is
equal to 28. Taking into account possible dropouts, 32 people for supplementation with the
test product and 32 people for supplementation with control product for a total of 64
volunteer subjects in this study.
The assessment is made on the basis of the abundance of the decrease of LDL cholesterol in
plasma, on the basis of the measures (mean and standard deviation) of LDL cholesterol
reported in a parallel study published in the European Journal of Nutrition. The parameter
LDL cholesterol, in addition to being an effect on human health recognized is more
restrictive and requires observation times longer than the extent of the modification of the
intestinal microflora.
Direct access to source data / documents Regulatory authorities may wish to carry out checks
on clinical research to evaluate compliance with the principles of GCP. Regulatory
authorities may also wish to conduct an inspection (during the trial or after its
completion). If an inspection required by a regulatory authority and / or the Ethics
Committee for Clinical Trials c/o APSS Trento, the researcher must agree to provide access,
regulatory authorities, the documentation related to the study.
Data Management and Records The source of the data and documentation is defined as the first
place where the data is recorded. Any original documents and should not be kept and made
available at the same location. The researcher must keep the original documents for each
person participating in the study.
The data collected on the CRF during the trial will be documented in an anonymous way, and
those will be identified by numbers or symbols. If in exceptional circumstances it is
necessary, for reasons of safety or regulation, identifying the person, OlioCRU Ltd and the
researcher are required to keep such information confidential.
All data entered in the CRF shall be legible and recorded in black ink. Should you require a
correction it should be made by deleting the entry with a single line and entering the
correct information next to the item itself. The correction must be initialed and dated by
the investigator, or a qualified person designated.
