Study design
To formally validate if APOE isoform, formed by rs7412 and rs429358, and CYP7A1 rs3808607 can predict responsiveness to PS consumption across the general population, the present proposal is to carry out a clinical trial at a) the University of Manitoba’s Richardson Centre for Functional Foods and Nutraceuticals (RCFFN) and b) Seven Oaks General Hospital (SOGH) in Winnipeg, Manitoba, Canada. The trial will use a priori recruitment of 64 individuals (Table 3) with the specific SNPs associated with responsiveness to PS. The present trial will therefore precisely select individuals from the general population with specific SNPs and then test their responsiveness to PS consumption. These responsiveness characterizations will generate the required data to validate the genoset-based classifications of responders and non-responders.
The clinical trial will follow a double blind, placebo-controlled, randomized two-period crossover study to investigate the LDL-C responsiveness to controlled administration of PS. The PS treatment will consist of two daily single portions of margarine, providing 1 g each of PS during the PS period (2.0 g/day of PS in total). The placebo treatment will be an identical margarine, except it will not contain any added PS. Both the PS and placebo margarine treatments will be anonymously coded by the industrial partner organization, Unilever, and provided to the research group to maintain blinding of both the researchers and participants throughout the clinical trial.
Each treatment period will consist of 28 days, with a minimum of washout of 21 days between periods. Participants will be required to attend breakfast at the RCFFN or Seven Oaks General Hospital (SOGH) and consume a meal containing one daily portion of margarine under supervision from Monday to Friday; the additional daily portion will be consumed with their evening meal. Participants will be given their evening and weekend margarine portions to take home for consumption. Participants will be provided diaries in which they are instructed to record when they ate the margarine in the evenings and on the weekends. During the week, participants will be required to return the empty margarine tubs on the following day to help monitor compliance, with margarine tubs used on Saturday and Sundays’ being returned on Monday. The return of the empty tubs and the confirmation of recording the consumption in the study diaries will be verified by clinical coordinators using a compliance checklist. Additionally, serum non-cholesterol sterols, including sitosterol and campesterol, the two main PS in the margarine, will be measured to monitor compliance. It is thought that the partial supervision of the treatments and monitoring of non-supervised treatments allows for a balance between compliance and participant burden.
Compliance will be further monitored through the visual confirmation by clinical coordinators that participants consumed the morning treatment on weekdays under supervision. Missed treatment consumption and return of margarine tubs will be recorded for each participant. Non-compliance will be defined as 1) missing supervision, 2) if participants fail to return at least 80% of the total empty margarine tubs per treatment period, and 3) missing 2 consecutive measurements or blood sampling days. Non-compliant participants will be asked to leave the trial; however, they will be compensated pro-rated based on the duration that they were involved in the trial.
Additionally, on a weekly basis, clinical coordinators will ask participants to report any changes in diet, lifestyle (sleep) or physical activity which may interfere with results of the trial and any other health outcomes or symptoms they may experience during the trial. Fasting blood samples are collected from participants on two consecutive days at the beginning (Days 0 and 1) and at the end (Days 28 and 29) of each trial period as described in the Table 4.
Study participants
Participants, 64 in total, will be recruited using various established methods, including flyers around the University of Manitoba and Seven Oaks General Hospital (SOGH), newspaper advertisements, direct mail advertisements within the City of Winnipeg, and advertising digitally at the Active Living Center of University of Manitoba as well as SOGH social media, websites and newsletter advertising among 6500 members of SOGH. An internal list of previous volunteers who have expressed interest in participating in other clinical studies will also receive an advertisement. Participants will be initially screened for eligibility over the telephone by the study coordinator if they respond to advertisements. If eligible, potential participants will be invited to the clinical research unit at the RCFFN or SOGH for an information session to introduce the research staff and provide further information about the study. Those expressing further interest will be invited to consent to and have a blood sample taken to ensure they meet all other trial criteria as listed below. Blood samples will be taken by RCFFN or SOGH phlebotomists or registered nurses. Screening blood samples are analyzed for the following: fasting lipid profiles including LDL-C, total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), and triglyceride (TG) concentrations, as well as, glucose, serum creatinine, blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), serum total protein, and serum albumin; all will be measured using the automated enzymatic methods on the Cobas 311 analyzer (Roche Diagnostics GmbH, Mannheim, Germany) or measured by Diagnostic Services Manitoba (DSM) according to their standard protocols. DNA will be extracted from blood sample buffy coat using commercially available column-based DNA extraction kits (DNeasy Blood and Tissue Kit, QIAGEN Sciences) according to the manufacturer's instructions. The concentration and integrity of the genomic DNA will be assessed by micro-volume spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific). DNA samples will then be genotyped by TaqMan SNP genotyping assays (CYP7A1-rs3808607, assay identification (ID) C2749212120; APOE rs7412, assay ID C2749212120; APOE-rs429358, assay ID C308479320; Life Technologies) on a StepOnePlus Real-Time PCR System (Applied Biosystems; Life Technologies). Data from the screening blood sample will be used to screen participants based on the predefined inclusion and exclusion criteria.
Inclusion criteria
Participants, men and women, aged 18 – 70 years with LDL-C concentrations of 3.0-4.9 mmol/L will be recruited into the trial. Participants must have a fasting glucose concentration of <6.1 mmol/L. A prospective recruitment scheme based on genotype will recruit 20-22 individuals in each of the three most common combinations, also called genosets, (outlined in Table 3). This would leave a minimum sample size of 20 participants for each individual genotype. The prospective recruitment based on genosets of interest will require screening of 200-400 potential participants. Such lengthy screening is required to find sufficient individuals who meet all the inclusion and exclusion criteria with the rarer genosets. Additionally, participants must be willing to: fast 10-12 hours before blood sampling; abstain from alcohol two days prior to blood sampling; and abstain from coffee and physical exercise for at least 12 hours before measurements and blood sampling. All participants must be able and willing to give informed consent to participate in the trial prior to their inclusion.
Exclusion criteria
Participants will be excluded if they are consuming, or have consumed in the last 3 months, medications or nutritional supplements which are known to affect lipid metabolism (such as cholestyramine, colestipol, niacin, clofibrate, gemfibrozil, probucol, HMG-CoA reductase inhibitors (statins), methotrexate, high-dose dietary fiber supplements, or plant sterols or stanols), or have any dietary restrictions which would prevent them from consuming the trial treatments. Participants who have a BMI >40 kg/m2 will be excluded. Participants must not have self-reported weight gain or loss greater than 3 kg in the past three months. Participants must be free of active cardiovascular disease including stroke, congestive heart failure, myocardial infarction, unstable angina pectoris, coronary artery bypass graft, percutaneous transluminal coronary angioplasty, temporal ischemic attacks, anemia, abnormal electrolytes, proteinuria, and abnormal liver, kidney, or thyroid function. Participants will be excluded if they have clinically significant biochemistry defined as: LDL-C <3.0 mmol/L or >4.9 mmol/L; TC > 6.2 mmol/L; fasting glucose > 6.1 mmol/ L, fasting TG >4.52 mmol/L; AST >100 U/L; ALT >100 U/L or any other clinically significant abnormalities in hematology and/or biochemistry at the investigator’s discretion.
Participants will be excluded if they have phytosterolemia, type 1 or type 2 diabetes, a history of cancer or malignancy in the last 5 years, or any metabolic disease, gastrointestinal disorder, or other clinically significant disease/disorder which could interfere with the results of the study or the safety of the participant. Participants will be excluded if they are smokers, tobacco/snuff/nicotine users, recreational drug users, or if they consume more than 14 alcoholic beverages a week. Participants who are pregnant or plan to become pregnant during the trial period will be excluded. Lactating women will also be excluded. Patients with unstable or serious illness, for example, dementia, terminal illness, recent bereavement, recent significant medical diagnosis will also be excluded. Employees of Unilever, Nutritional Fundamentals of Health (NFH) and the research institutes conducting the research will not be allowed to participate in the study.
Randomization
Eligible participants will be randomly allocated to two groups: the PS treatment group or the placebo group for the first period and then participants will switch treatments for the second period after the washout between periods. Randomization will be done by an assistant outside of the research team using a block randomization method through sealed envelopes with stratification by sex and genoset. Randomization in blocks of eight and four, each with equal numbers of treatment orders will be used. This blocking is being done to minimize imbalances in treatment orders within each genoset group or by sex. Administration of the intervention will be conducted in a double-blind manner. Single portion tubs of PS treatment and placebo margarine are being created for this study by Unilever and are being delivered to the research team in identical packages labeled either A or B.
Remuneration
Study participants will receive up to a total of CAD $400 (i.e., $200/period x 2 periods) for study completion. This amount will be divided into 2 portions. Participants will receive $200 after completion of period 1 and another $200 after completion of period 2. If a participant withdraws early from the study, they will receive an appropriate pro-rated fraction of this amount.
Outcome measures
Primary outcome
Serum LDL-C concentration and its change in response to PS consumption, is considered the primary outcome of this trial. Approximately, 20 mL blood samples on days 0, 1, 28 and 29, of the intervention period will be collected. Serum lipid profile (TC, LDL-C, HDL-C, and TG) will be measured using the Cobas 311 analyzer (Roche Diagnostics GmbH, Mannheim, Germany). Average values of days 0 and 1 will be used as baseline, and average of days 28 and 29 will be endpoint values.
Secondary outcomes
At baseline and at the end of the two intervention periods, anthropometric measurements including body weight, BMI, hip and waist circumference, and blood pressure will be measured. Blood pressure will be measured in an office setting on. Days 0, 1, 28, and 29 of each treatment period. Participants will ask to rest 10 minutes prior to having their blood pressure taken, in the event they rushed in. This measurement will take place in a quiet room while the participant is in a seated position and arm rested on an armrest at heart level. Participants will be advised to rest quietly throughout the measurements. Blood pressure measurement will be performed four times at 2-minute intervals. Gastro-intestinal tolerability questionnaires will be completed by participants at the beginning and at the end of each intervention period. The 10-year CVD risk score will be calculated for each participant during each intervention period utilizing the ACC/AHA Cholesterol Guideline risk calculator. Participants will be asked to complete a questionnaire upon completing the trial which asks them whether they think they know which treatment they received during with treatment period. This information will be used to verify participant blinding.
Fasting serum glucose, will be measured with the Cobas 311 analyzer (Roche Diagnostics GmbH, Mannheim, Germany). Plasma samples will be used to quantify concentrations of blood sterols and sterol precursors (non-cholesterol sterols, NCS) according to previously established method (8). Authenticated internal standards will be added to plasma samples, which will then be saponified with methanolic KOH solution. Sterols will then be extracted twice with petroleum ether. Extracted sterols will be derivatized using a tri-methylsylation (TMS) procedure. The TMS-derivatized samples and sterol analysis will be carried out by gas chromatography with flame ionization detection. Campesterol, sitosterol, campestanol, sitostanol, and cholestanol, as well as lanosterol, desmosterol, and lathosterol will be measured.
Fractional cholesterol synthesis will be measured by deuterium incorporation according to previously established procedures (7, 10, 11). 24 hours before the end of each treatment period participants will be asked to consume deuterium water (D2O) given at a dose of 0.7 g/kg body water (estimated at 60% of total body weight). D2O is a stable isotopic tracer and poses no radiation hazard and can be safely administered to human participants. D2O water will be administered orally. A fasted blood sample will be taken at baseline and on day 28 prior to isotope administration, as well as fasting samples on day 29. The change in deuterium enrichment within red blood cell free cholesterol will be determined as an index of cholesterol synthesis over days 28 and 29.
Sample size calculation and statistical analysis
The sample size, with a minimum of 20 participants for each individual genotype, and n=20-22 for each individual genoset is based on previous work performed by this research group. A power calculation was performed using PROC POWER (a = 0.05 and b = 0.80; SAS Institute (version 9.4)) based on an average reduction in LDL-C of 0.34 mmol/L and a variance of SD = 1.05 mmol/L resulting from PS consumption according to the meta-analysis findings of Demonty et al. (12).
Given the crossover design the study outcomes measures will be analysed in a per-protocol population where only participants who received both treatment and placebo are included. The effects of treatment, comparing the endpoint values of the treatment and placebo periods, will be analyzed by the SAS MIXED procedure. Sequence and sex will be included in the model as fixed factors, while participant will be included as a random and repeated factor. Genoset and treatment by genoset will be included as fixed factors to assess the impact of genoset on treatment. The impact of the individual genotypes will also be investigated individually. Significant treatment-by-genoset or treatment-by-genotype effects will be examined by the SAS SLICE function, with Bonferroni correction for the number of slices. Treatment effect sizes by genoset or genotype, from significant interactions, will be compared by t test or ANOVA using the difference in mixed-model least squares means summary statistics for the treatment effect slices, with Tukey-Kramer adjustment for multiple comparisons (10).