EFFECTS OF “FOODS FOR HEALTH” PORTFOLIO OF FUNCTIONAL FOOD PRODUCTS ON LIPID MANAGEMENT IN INDIVIDUALS UNWILLING TO USE STATIN THERAPY

ABSTRACT

Dietary solutions can ameliorate cardiovascular disease (CVD) risk in statin reluctant patients. Yet no studies have been undertaken to explore if a portfolio approach with healthy appetizing ready-to-eat foods having functional food ingredients can improve cardiovascular health in this population. The present study primarily examines the effect of a range of proprietary products containing four functional bioactives, on cholesterol levels in statin reluctant participants. A multi-center, randomized, double-blind, free-living cross-over study composed of 2 regimented phases of 4 weeks each, separated by 4 weeks of washout was conducted. Participants (n=54) ingested two servings per day from an assortment of packaged, shelf-stable food products as a substitute for some foods they were already consuming. Treatment products consisted of oatmeal, pancakes, cranberry bars, chocolate bars, sprinkles, and smoothies formulated specifically to provide 5g of fiber, 1000 mg of alpha-linolenic acid, 1000 mg of phytosterols and 1000 μmol antioxidants per serving. Control products were calorie-matched like items drawn from the general grocery marketplace. Fasting glucose, triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC) concentrations and low-density lipoprotein cholesterol (LDL-C) were analyzed at baseline and endpoint of both phases. Ingestion of foods was confirmed by plasma fatty acid profiling at the endpoint of both phases for Alpha Linolenic acid (ALA) levels. CYP7A1-rs3808607 and APOE heterogeneity were analyzed for associations with variability in LD-C responses. A reduction of 5.08±8.24 % was observed in total cholesterol (p-value 0.0001) with LDL-C levels being reduced by 8.80±12.5 % (p-value <0.0001) after consumption of study foods compared to control. Circulating TG levels, HDL-C, and glucose concentrations were not influenced by study foods. The results reveal that consumption of a portfolio of ready-to-eat bioactive-containing foods significantly improves serum lipid profiles in patients unable or unwilling to take statin drugs. This novel, easily adaptable food-based approach is anticipated to have extensive implications for healthcare research and practice improvement.

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ACKNOWLEDGEMENTS

DEDICATION

TABLE OF CONTENTS

ABSTRACT

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ACKNOWLEDGEMENTS

DEDICATION

LIST OF TABLES AND FIGURES

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LIST OF ABBREVIATIONS

1.BACKGROUND AND INTRODUCTION

1.1 INTRODUCTION

1.2 ROLE OF FUNCTIONAL FOODS IN HYPERLIPIDEMIA MANAGEMENT

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2. LITERATURE REVIEW

2.1 PHYTOSTEROL

2.2 FIBER

2.3 ALPHA-LINOLENIC ACID (ALA)

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2.4 ANTIOXIDANTS

2.5 PORTFOLIO OF FUNCTIONAL FOODS

2.6 HETEROGENEITY IN CHOLESTEROL RESPONSE ACROSS INDIVIDUALS

3. RATIONALE, HYPOTHESIS AND OBJECTIVES

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3.1 RATIONALE

3.2 HYPOTHESIS

3.3 OBJECTIVES OF THE RESEARCH

4. MANUSCRIPT

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4.1 ABSTRACT:

4.2 INTRODUCTION:

4.3 EXPERIMENTAL METHODS:

4.3.1 STUDY DESIGN

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4.3.2 ETHICAL CONSIDERATIONS

4.3.3 PARTICIPANT SELECTION CRITERIA

4.3.4 PARTICIPANT RECRUITMENT AND FLOW

4.3.5 OUTCOME MEASURES

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4.3.6 STATISTICAL ANALYSIS

4.4 RESULTS:

4.4.1 PARTICIPANT CHARACTERISTICS:

4.4.3 ANTHROPOMETRICS AND BLOOD PRESSURE:

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4.4.4 BLOOD LIPIDS:

4.4.5 GLUCOSE, INSULIN AND HSCRP:

4.4.6 FATTY ACIDS:

4.5 DISCUSSION:

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4.6 CONCLUSION:

5. GENERAL DISCUSSION AND CONCLUSION

6. STRENGTHS AND WEAKNESSES OF THE STUDY

7. REFERENCES

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8. APPENDICES

8.1 ETHICS APPROVAL FORMS

8.2 RESEARCH PARTICIPANT INFORMATION AND CONSENT FORM…

8.3 ADDITIONAL RESEARCH PARTICIPANT INFORMATION AND CONSENT FORM FOR GENETICS ANALYSIS

8.4 STUDY ADVERTISEMENT POSTER

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8.6 WEEKLY CALL SCRIPTS

8.7 THE AMERICAN HEART ASSOCIATION’S DIET AND LIFESTYLE RECOMMENDATIONS

8.8 PRODUCT USE INSTRUCTION SHEETS

8.9 PRODUCT ACCOUNTABILITY LOGS

8.10 CASE REPORT FORM

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LIST OF TABLES AND FIGURES

Table 1. Summary of key nutrients and ingredients in the study food products

Table 2. Summary of trial plan

Table 3.  Baseline characteristics of study participants

Table 4. Summary of side effects at the end of the treatment phase

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Table 5. Endpoint analyses of cardiovascular risk factors in response to study food consumption compared to control

Table 6. Baseline-to-endpoint analyses of cardiovascular risk factors in response to study foods compared to control

Table 7. Endpoint analyses of plasma fatty acid profile in response to study foods compared to control

Table 8. Serum LDL-C responsiveness to study foods compared to control categorized by SNPs

Figure 1. Participant flow chart

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Figure 2. Percentage differences in lipid profile

Figure 3. Waterfall Plot of Percentage LDL-C Differences in Individuals Between Treatment and Control Foods

Figure 4. LDL-C differences stratified by Apo E isoform

Figure 5. LDL-C differences stratified by CYP7A1 genotype

LIST OF ABBREVIATIONS

ACAT- 2 -Acyl-coenzyme A cholesterolacyltransferase-2

AHA – American Heart Association

ALA – alpha-linolenic acid

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APOE – Apolipoprotein E

CVD – Cardiovascular disease

CYP7A1 – Cholesterol 7 α-hydroxylase

DHA – Docosahexaenoic acid

EFSA – European Food Safety Authority

EPA – Eicosapentaenoic acid

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FDA – Food and Drug Administration

HDL-C – High-density lipoprotein cholesterol

HMG Co-A reductase – 3-Hydroxy-3-methyl-glutaryl-coenzyme A reductase

LDL-C – Low-density lipoprotein cholesterol

LXR – Liver X receptors

MTP – Microsomal triglyceride transfer protein

NCEP – National Cholesterol Education Program

PUFA – Polyunsaturated fatty acids

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SNP – Single-nucleotide polymorphisms

TC – Total cholesterol

TG – Triglyceride

TICE – Trans-intestinal cholesterol excretion

VLDL – Very Low-density lipoprotien

1.BACKGROUND AND INTRODUCTION

1.1 INTRODUCTION

Cardiovascular diseases (CVDs), the leading avertible cause of death in the world accounts for 17.3 million deaths per year according to the health statistics report 2015 by American Heart Association(AHA).^1,2 The improvement of cardiovascular health is thus a global necessity and the AHA points out ‘control of cholesterol’ as one of the seven key health factors to move towards this goal.³ The underlying disease process of CVD is mainly ‘atherosclerosis’ which find its path through behavioral risk factors including less physical activity and unhealthy diet rich in fat and calories. The metabolic risk factors such as elevated blood lipids and hypertension aggravates the problem leading to cardiovascular disease conditions.² Dietary solutions can play a potential role in modifying lipid profile and thus ameliorate CVD risk factors.^4,5

1.2 ROLE OF FUNCTIONAL FOODS IN HYPERLIPIDEMIA MANAGEMENT

Dyslipidemia, characterized by alterations in serum lipoprotein levels, is one of the major culprits of atherosclerosis and chronic heart disease.⁶  This is supported by the fact that a mere 1 mmol/l (39.5 mg/dL) decrease in the low-density lipoprotein cholesterol (LDL-C) level reduces the rate of coronary events by 20% and total mortality by 12%, regardless of baseline risk.⁷

Statins constitute a major class of cholesterol-lowering medications that act by inhibiting HMG Co-A reductase (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase), the rate-controlling enzyme of cholesterol synthesis in the body^8,9 , and have excellent data enough to place them as first-line drug of choice for patients with hypercholesterolemia, mixed hyperlipidemia and a history of cardiovascular disease.7 However, 10-30 % of patients treated with statins suffer from muscle-related side effects in a dose-dependent manner.¹⁰ These side effects range from myalgia to myositis and rhabdomyolysis, that can result in acute renal failure and ultimately death. Studies also reveal that statins can lead to an increased risk of diabetes mellitus (10–12 %). These side effects result in statin intolerance in certain population creating hindrance against the preventive goal targets for CVD.⁷

Availability of a conveniently formulated functional food combination aimed at reducing LDL-C can be utilized especially by four categories of individuals who are at risk of developing CVDS which include individuals who are statin intolerant, individuals who are reluctant to take statin due to other reasons individuals who are unable to achieve LDL-C targets at maximum tolerated dose of statin and individuals who are unable to respond to statins.^11,12

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The well studied functional food ingredients; phytosterol, fiber, omega -3- fatty acids and antioxidants, could play a key role in creating a diet conducive to heart health that could be effectively utilized by this population. These functional foods are extensively documented in the literature for their beneficial role in cholesterol levels individually, and are discussed in the literature review section.^6,12 There are only a limited number of studies looking into portfolio hypocholesterolemic effect of nutritional bioactives which are also outlined in the following literature review section.¹³

Alike medications, the functional food bioactives also utilize cholesterol metabolism pathways to exert control over circulating cholesterol and thus on CVD risk. A marked inter-individual variability in cholesterol response, linked to heterogeneity of metabolism pathways, has been evidenced by observational and dietary intervention studies.¹⁴ Two single-nucleotide polymorphisms (SNPs) are repeatedly found to be associated with variability in response to phytosterol and or fiber consumption which are also briefly discussed in the literature review section.

2. LITERATURE REVIEW

2.1 PHYTOSTEROL

Phytosterols refers to plant-derived sterols and stanols, compounds that are structurally similar to cholesterol. Beta-sitosterol, campesterol, and stigmasterol are the major sub-classes of the plant-sterol family whereas its saturated forms; sitostanol, campestanol and stigmastanol represent the stanol group.^15,16

Phytosterols/stanols are well recognized cholesterol-lowering agents that facilitate primary and secondary prevention of cardiovascular diseases.¹⁷ This is underlined by the fact that various national and international guidelines such as The American Heart Association ¹⁸ and the European Atherosclerosis Society consider phytosterols as an effective treatment option for lowering cholesterol.¹⁹ According to these guidelines, a daily addition of 2 g/day plant sterols or stanols as part of a healthy diet can reduce CVD risk in subjects with elevated LDL-C concentrations. Up to 10% reductions in LDL-C can be achieved through this approach.²⁰

The mechanism by which phytosterols/stanols exert its cholesterol-lowering effect is due to its similarity to cholesterol in structure and metabolic pathway. Dietary cholesterol undergoes cleavage from esterified into free form and get solubilized in the lumen by the action of bile acids; incorporated into mixed micelles and penetrate through mucosal barriers such as the unstirred water layer and the brush border membrane and are absorbed into enterocytes through Niemann-Pick C1-like transporter. After absorption into the enterocyte, cholesterol is re-esterified by intestinal enzyme, acyl-coenzyme A cholesterolacyltransferase-2 (ACAT-2). These cholesteryl esters are incorporated into chylomicrons through microsomal triglyceride transfer protein (MTP) and are secreted into the lymph.

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Phytosterols exhibit competitive inhibition in this pathway, primarily by displacing cholesterol from mixed micelles. Unlike cholesterol, after absorption into enterocytes, phytosterols remain in free form itself being poor substrates for ACAT-2. This interference in cholesterol absorption results in excretion of unabsorbed cholesterol through faeces.^11,21

Competition for incorporation into chylomicrons, competition of the involved transporters, and activation of liver X receptors (LXR) target genes are the other suggested theories. Recently a trans-intestinal cholesterol excretion (TICE) pathway theory has also been postulated as a rationale for the cholesterol-lowering action of the plant sterols and stanols. Stimulation of TICE accelerates faecal sterol loss.²⁰

 2.2 FIBER

Dietary fibers are large group of bioactive carbohydrate polymers and include naturally occurring; edible as well as extracted and synthetic carbohydrate polymer.⁶ Consumption of dietary fibers found in oats, barley, and psyllium have been shown to reduce total cholesterol (TC) and LDL-C levels at statistically significant levels, which is scientifically substantiated by epidemiologic studies, randomized clinical studies and subsequent meta-analyses. Most studies could not establish any advantageous effect of fiber consumption on triglyceride (TG) or High-density lipoprotein (HDL) cholesterol levels.^12,22,23

Dietary fiber, in general, is classified into two categories i.e. water-insoluble/less fermented fibers; cellulose, hemicellulose, lignin and the water-soluble/well-fermented fibers; pectin, beta glucan, and mucilage.

Water-soluble fibers are important in imparting a hypocholesterolemic effect as evident through clinical studies and which are in turn supported by the proposed mechanisms by which fiber lowers cholesterol. A major proposed mechanism is that water-soluble fibers interfere with the absorption of dietary fat and bile acid cholesterol and enhance its excretion through feces. This might be through creating a barrier on intestinal surface hindering cholesterol absorption, entrapment of cholesterol in its own local matrix and or, directly binding to cholesterol molecule. In addition, soluble fiber absorbs water in gastrointestinal tract resulting in increased bulk of stools and decreases the intestinal transit time. This can have advantageous effects on satiety by sustaining a ‘fullness’ feeling. Another proposed theory is that soluble fiber hinders the absorption of glucose and nutrients which will, in turn, result in a low glycemic response and thus, a decline in insulin level. The hormone insulin has a significant role in the synthesis of hepatic cholesterol through activation of HMG-Co A reductase enzyme. Thus, a low insulin level can slow down hepatic cholesterol synthesis. An additional theory is based on colonic fermentation of soluble fiber resulting in short chain fatty acids, especially propionate that can hinder hepatic cholesterol metabolism through various pathways.

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The latter two mechanisms are less supported as a dominant mechanism of fiber action. On the other hand, the only effect water-insoluble fiber can exert in this scenario is that they substitute foods containing saturated fats and cholesterol.^24,25

Although there are studies showing a cholesterol-lowering effect of fibers from 3g per day consumption; 10g/day is the least recommended dose to attain a clinically significant reduction of 3-5% in LDL-C levels.¹²

There are numerous studies pertaining cholesterol-lowering effects of fiber.^24,26,27 However, the advantages of this ingredient as a functional food onside with other major functional food actives that can exert a similar effect through other mechanisms require further studies in order to reveal any beneficial effects it can have on cholesterol lowering.

2.3 ALPHA-LINOLENIC ACID (ALA)

Dietary polyunsaturated fatty acids (PUFA) can be divided into n-6 (or omega-6) and n-3 (or omega-3) PUFA. Two important n-6 PUFA are linoleic acid and arachidonic acid. Major n-3 PUFA are eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and are present abundantly in fish, shellfish, and sea mammals.²⁸ EPA and DHA are synthesized by phytoplankton which are microscopic water-plants, and primary producers of marine life. On the other hand, plants of the land also offer a rich source of n−3 PUFA, the 18-carbon ALA.²⁹

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ALA is an essential fatty acid and is present in flaxseed, soybean, canola, and in walnuts.³⁰ Humans can convert ALA into EPA and DHA and may also confer additional health benefits by itself. ²⁹  The conversion efficiency of ALA to EPA varies between 0.2%-21% average being 8%, and that of ALA to DHA ranges between 0%-9% with average being <1%, and affected by multiple factors.²⁸

Epidemiologic studies support use of ALA to prevent arrhythmias (ventricular tachycardia and fibrillation). The n-3 PUFA have anti-inflammatory and antithrombotic properties. Further, these FA may stimulate endothelial-derived nitric oxide, and hence are vasoprotective. In addition, they have hypolipidemic properties with effects on triacylglycerols and very Low-density lipoproteins (VLDLs), and inhibit atherosclerosis.²⁹ A meta-analysis by Pan et al. (2012) reviewed 27 dietary and biomarker studies relating ALA to CVD events and concludee that there is 10% lowered risk of death from chronic heart disease with each 1 g/day intake of ALA. ³¹. Thus, ALA is potential functional ingredient present in plant sources that can be used in a portfolio diet for CVD risk prevention.

2.4 ANTIOXIDANTS

The advantages of functional food compared to supplements are that functional foods can confer additional benefits such as due to presence of anti-oxidants. Antioxidants can beneficially interfere with diseases-related oxidative stress and modify cardiovascular risk factors. There are variety of targets and mechanisms of action of antioxidants conferring to cardiovascular benefits, which includes anti-oxidative and anti-inflammatory effect, autonomic adrenergic response modulation and endothelial function modification.^32–36

 2.5 PORTFOLIO OF FUNCTIONAL FOODS

David Jenkins is the pioneer who introduced portfolio diet elucidating the beneficial effects of combining plant sterols, soy protein, viscous fibers and almonds in modifying lipid profile.^37–42 There are five portfolio studies based on this diet which are discussed below.

The first portfolio study in 2002 hypothesized the additive effect of the portfolio diet.  This was almost a full feeding study except for vegetables and low-calorie fruits, with 13 participants. The intervention was 4 weeks with a diet low in saturated fat and high in plant sterols (1 g), soy protein (22.7 g), and viscous fibers (8.2 g), almonds (2.9g) /1000kcal sourced on foods obtained from supermarkets and health food stores. There was a substantial reduction of 29% in LDL-C matching efficacy of statins. However, the study used only historical control readings and no control arm, the effect of low-fat diet was not assessed, and food acceptability was scored only 6.3/10; which meant “acceptable with minor modifications”.  In addition, food was reported as very filling having 9 out of 13 willing to continue diet with minor modifications.^43–45

In 2003 Jenkins et al. conducted another study to ascertain the effect of portfolio low-fat diet modifying the study design to include a control arm. There were 12 versus 13 participants in control and study arm of the parallel open labelled study, respectively.  The diet utilized same ingredients although there were some variations in amount being very low in saturated fat and high in plant sterols (1.2 g), soy protein (16.2 g), viscous fibers (8.3 g), and almonds (16.6 g) /1,000 kcal. There was a 2-week pre-wash period to get rid of any previous statin effects that participants were consuming. The study demonstrated an LDL-C reduction of 35% treatment arm and 12.1% in control arm. The low sample size and complex diet were the major drawbacks of the study.^43,46,47

Further in 2005, the group reported a study giving a direct comparison of the portfolio to statin. The study used a low-fat control diet, low fat plus statin diet and the portfolio diet. It was an open labeled study of crossover design and the diet was very low in saturated fat and high in plant sterols (1.0 g), soy-protein foods (21.4 g), almonds (14 g), and viscous fibers (10 g) /1000 kcal. The study diet showed a 29.6% reduction in LDL as compared to 33% with statin. Although the effect was less than that of statin along with low-fat diet, a greater number of participants were able to achieve their target concentrations through consumption of the portfolio diet in the study.⁴⁸

Another long-term study by Jenkins in 2006 demonstrated a self-selected portfolio with a duration of 1 year. The study reported a 12% LDL-C reduction which was lower than expected, for which low compliance was a major reason. Although 55 participants completed the study, only 26 participants showed greater than 55% compliance.^7,11,12

Further, a portfolio study in 2011 used this diet at 2 levels of intensity; routine portfolio and intensive with 2 and 7 clinic visits for dietary counseling respectively, over a period of 6 months. The study reported no additional benefits with intensive visit portfolio having 13.8% LDL-C reduction as compared to 13.1% reduction observed with routine portfolio. The study was not metabolically controlled and helped to mimic real world effects. However, the dropout rate observed in the study was 22.6%.^3,48,49

In summary, portfolio studies have shown significant beneficial effects on lipid profiles suggesting advantages of combining functional ingredients to reduce CVD risk. However, this dietary pattern requires a strong commitment that resulted in poor compliance in all these studies. Further, there are no studies examining the effect of such a combination in statin reluctant people. A portfolio of ready to eat healthy, tasty foods that can confer the beneficial effects of fiber, phytosterol, Omega -3 fatty acids and anti-oxidants might be able to accomplish cardiovascular risk reduction in a patient population that cannot tolerate statins.

2.6 HETEROGENEITY IN CHOLESTEROL RESPONSE ACROSS INDIVIDUALS

Although there are genetic variants that have been associated with phytosterol response, the available data on gene-response associations are mostly inconsistent. There are a few single-nucleotide polymorphisms that are linked with variability in cholesterol response to phytosterol consumption and only two of them have been replicated by interventional studies so far. These are rs3808607 in cholesterol 7 α-hydroxylase (CYP7A1) and apolipoprotein E (APOE) isoform. De Castro-Oros et al. (2011), for the first time, demonstrated that rs3808607 in the promoter region of the CYP7A1 gene is linked with response to plant sterol consumption. MacKay et al. (2015) replicated this finding and concluded an allelic dose effect of CYP7A1 with T/T carriers giving least response and an upsurge in response with each G-allele. In addition, Wang et al. (2016) found a similar association of the SNP in LDL-C lowering by a high molecular weight soluble fiber of β-glucan.^48,50–52

Miettinen and Vanhanen (1994) and Vanhanen et al. (1993), found an improved cholesterol lowering with phytosterol consumption in APOE4 carriers compared to E2 and E3, which was also replicated by MacKay et al. (2015). These findings suggest the possibility of predictable cholesterol responses to functional actives affecting cholesterol metabolism; with the aid of these SNPs.^38,53

3. RATIONALE, HYPOTHESIS AND OBJECTIVES

3.1 RATIONALE

Despite the recognition that dietary interventions can ameliorate cardiovascular disease risk in statin reluctant patients, there have been no studies conducted that explore the use of a portfolio of healthy appetizing foods in improving health in this population.

3.2 HYPOTHESIS

It was hypothesized that a portfolio of recommended ready to eat healthy foods can improve serum lipid profiles and reduce markers of cardiovascular disease (CVD) risk in a patient population who are unable to tolerate a medication-based approach for risk factor reduction.

3.3 OBJECTIVES OF THE RESEARCH

The overall objective of this project was to, investigate the ability to affect CVD risk factors in a statin reluctant population through a novel, easily implemented functional food-based approach. The specific objectives were:

• To evaluate the effect of consuming a range of specifically formulated proprietary products on serum low-density lipoprotein (LDL)-cholesterol, triglyceride (TG) glucose, insulin, high-sensitivity C-reactive protein (hsCRP) concentrations and on plasma fatty acid profile.
• To associate potential SNPs affecting cholesterol metabolism with the level of LDL-responsiveness.

4. MANUSCRIPT

Effect of portfolio functional food products on lipid management in individuals reluctant to statin therapy

Soumya Alias^1,2, Jessica Bauman³, Stephanie Jew², Elizabeth Klodas³, Stephen Kopecky³, Peter J. H. Jones^1,2

¹Department of Food and Human Nutritional Sciences, University of Manitoba, Winnipeg, MB, Canada, ²Richardson Centre for Functional Foods and Nutraceuticals, Winnipeg, MB, Canada,³Mayo Clinic, Rochester, MN, USA

*Corresponding author: Peter J.H Jones

Department of Human Nutritional Sciences

Richardson Centre for Functional Foods and Nutraceuticals

196, Innovation Drive, SmartPark, Winnipeg R3T 2N2, Manitoba

Phone: (204)-474-8883; Fax: (204) 474-7552

Email ID: [email protected]

4.1 ABSTRACT:

OBJECTIVES: Dietary approaches can ameliorate cardiovascular disease risk in statin reluctant patients. Yet no studies have been undertaken to explore if a portfolio approach with healthy appetizing ready-to-eat foods having functional food ingredients can improve cardiovascular health in this population. The objective of our study was to evaluate the effect of a range of proprietary products, which contain four functional bioactives, on cholesterol levels in statin reluctant participants.

METHODS: A multi-center, randomized, double-blind, free-living cross-over study composed of 2 regimented phases of 4 weeks each, separated by a washout of 4 weeks, was conducted. Participants (n=54) ingested two servings per day from an assortment of packaged shelf-stable food products as a partial substitute for some foods they were already consuming. Treatment food products consisted of oatmeal, pancakes, cranberry bars, chocolate bars, sprinkles, and smoothies specially formulated in such a way to provide 5g of fiber, 1000 mg of alpha-linolenic acid, 1000 mg of phytosterols and 1800 µmol antioxidants per serving. Control products were calorie-matched like items drawn from the general grocery marketplace.

OUTCOME MEASURES: Fasting glucose, triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) concentrations were analyzed at the baseline and endpoint of both phases. Plasma fatty acid profiling was carried out at the endpoint of both phases to ascertain Alpha Linolenic acid (ALA) levels, to confirm the ingestion of study foods. CYP7A1-rs3808607 and APOE heterogeneity were analyzed for associations with variability in LDL-C responses.

RESULTS: A reduction of 5.08±8.24 % was observed in total cholesterol (p-value 0.0001) with LDL-C levels being reduced by 8.80±12.5 % (p-value <0.0001) after the consumption of study foods compared to control. Circulating HDL-C, TG levels, and glucose concentrations were not influenced by study foods.

CONCLUSIONS: Consumption of a portfolio of ready-to-eat bioactive containing foods significantly improves serum lipid profiles in patients who are unable or unwilling to take statin drugs. This novel, easily adaptable food-based approach is anticipated to have extensive implications for healthcare research and practice improvement.

4.2 INTRODUCTION:

Cardiovascular diseases (CVDs) remain as the prime cause of death worldwide and according to World Health Organization (WHO), at least 2 million premature deaths from CVDs could be prevented each year by achieving the risk-factor targets. ⁵³ Currently, HMG-Co A reductase inhibitors are the primary medications used in the prevention and treatment of CVDs. However, a marked population of those prescribed statins are reluctant for various reasons. This statin reluctance is due to the intolerance from side effects on muscles ranging from myalgia to myositis and rhabdomyolysis; and a growing concern about several other adverse effects including potentiating the risk for diabetes mellitus upon statin consumption. Further hypo-responders exist, who are unable to achieve their target goals with maximum dose of statin, and non-responders to statins. ^29,54,55

Lowering low-density lipoprotein cholesterol (LDL-C) is a key therapeutic approach for reducing the CVD risk and dietary therapy could play a vital role in this. Evidence-based guidelines of WHO, American Heart Association (AHA), as well as Canadian Cardiovascular Society recommend dietary modifications as a critical part of CVD risk reduction strategies.^32,33,36 Extensive research has been conducted through randomized clinical trials and cohort studies to determine the effects of individual dietary components on CVD risk factor reduction. ^43–45,56

A novel study based on plant-based dietary pattern termed as “portfolio diet” was introduced in the 2000s by Jenkins et al., which combined 4 core food components: nuts, plant protein, viscous fibre and plant sterols. All these ingredients have health claims for cholesterol-lowering or CVD risk reduction from Food and Drug Administration (FDA), Health Canada, and/or European Food Safety Authority (EFSA). The study was accompanied by further portfolio attempts with slight modifications in the ingredients and study designs re-enforcing its benefits on CVD risk. A recent meta-analysis pooled the existing 7 portfolio trials and concluded an additive effect of the portfolio ingredients with an average 17% LDL-C reduction being 21% in efficacy and 12% in effectiveness trials.

The Portfolio dietary pattern consisted of 4 core food components: nuts (42 g); plant protein (50 g); viscous soluble fiber (20 g); and plant sterols (2 g) (based on a 2000 kcal diet). All the 5 portfolio trials made use of background diet based on National Cholesterol Education Program (NCEP) Step II diet having limits of 7% energy from saturated fat, 30% energy from total fat, and 200 mg/day of cholesterol.  Thus, portfolio diet trials required the entire diet of participants fit these recommendations and results in a low overall compliance.^57,58 The feasibility of imparting such a marked dietary change and the commitment required warrants the need for further portfolio approaches that can be easily implemented and widely utilized.

In our study, two snacks per day substitution of shelf stable ready to serve foods, having commensurable amounts of plant sterols, soluble fibers, alpha-linolenic acid (ALA) and antioxidants, were utilized to provide a healthy appetizing basket of a portfolio approach. Systematic reviews and meta-analyses of randomized controlled trials have reported reductions in LDL-C on an average of 7% for soluble fibers at a consumption of 5–10 g/day and 7% for plant sterols when consumed 2 g/day.⁵⁹ ALA, the plant-based n-3 PUFA, undergoes conversion to icosapentaenoic acid (EPA) at a rate of 5–10% and docosahexaenoic acid (DHA) at 2–5% in the body. Epidemiologic studies and meta-analyses report that ALA may have hypolipidemic properties with effects on triglycerides (TGs) and very low-density lipoproteins (VLDLs) and can inhibit atherosclerosis.^50,53 Antioxidants, on the other hand, may confer cardiovascular benefits which include the anti-inflammatory effect, autonomic adrenergic response modulation and endothelial function modification.⁵³ Thus, combining these four ingredients might be able to exert added beneficial effects on CVD risk, especially within a statin reluctant population. To our knowledge, there were no other studies that explored if a portfolio approach with ready-to-eat foods having functional food ingredients can improve cardiovascular health in this population.

Generalized dose-response curves need to be redefined in this era of nutrigenetics in order to achieve personalized nutrition strategies. Clinical trials by De Castro-Oros et al. (2011), MacKay et al. (2015) and Wang et al. (2016) suggest varied cholesterol uptake and metabolizing abilities by CYP7A1-rs3808607 and APO-E variants. These studies demonstrated that CYP7A1-rs3808607 and APOE heterogeneity affects variability in response to phytosterol or fiber intake in LDL-C or TC lowering. Thus, it might be possible to predict the LDL-C responses of these functional ingredients to some extent by examining the role of CYP7A1 and APOE polymorphisms.⁵³

The objective of the present study was to examine the effect of a range of proprietary products containing four functional bioactives; plant sterols, soluble fibers, alpha-linolenic acid and antioxidants; on cholesterol levels in statin reluctant participants.  Our specific aim was to evaluate the changes in serum, High-density lipoprotein cholesterol (HDL-C), LDL-C, TG, total cholesterol (TC), glucose, insulin and high-sensitivity C-reactive protein (hsCRP) concentrations over 4 weeks regimen with the study foods. In this study, we also aimed to correlate the LDL-C responses as a function of CYP7A1 and APOE polymorphisms.

4.3 EXPERIMENTAL METHODS:

4.3.1 STUDY DESIGN

This dual-center study followed a randomized, free-living crossover design composed of 2 regimented phases of 4 weeks each, separated by 4 weeks washout. The clinical trial was conducted at the Richardson Centre for Functional Foods and Nutraceuticals (RCFFN), University of Manitoba, Winnipeg, Manitoba, Canada; and the Mayo Clinic, Rochester, Minnesota, USA.

Trial participants followed 4 weeks run-in period with their usual dietary patterns; maintaining their usual levels of physical activity throughout the study. A maximum of 2 alcoholic beverages per day was permitted during the study. After 4 weeks washout period, participants received packaged shelf-stable food products from Step One Foods, Truhealth Inc, presented in a single-serve format requiring minimal to no preparation. The treatment products consisted of foods such as oatmeal, pancakes, cranberry bars, chocolate bars, smoothies, and a sprinkle offering which can be added to almost any food to enhance its nutritional impact. All products were interchangeable in terms of their nutrients of interest and possessed a minimum of 5g of fiber, 1000 mg of omega-3 fatty acids, 1000 mg of phytosterols and 1800 µmol antioxidants per serving (Table 1). Calorie counts of the products ranged from 110-190 kcal per serving. Control products were like-items drawn from the general grocery marketplace. These products were identical in type, appearance and preparation requirements, and were matched for calories to the treatment products. Participants were instructed to consume 2 items over the course of each day as a substitute for breakfast, as a snack, or as a component of the main meal.

All participants received printed instructional materials outlining test product use and were verbally encouraged to follow the directions provided to them in the written materials. The participants were also contacted once a week over the course of intervention to encourage compliance. During intervention periods, participants were instructed to fill out a checklist of the number of products consumed. Compliance to the intervention protocol was determined using the completed checklists and food return counts. Medication use during the trial was also monitored. The trial plan is summarized in Table 2.

4.3.2 ETHICAL CONSIDERATIONS

The study procedures were approved by the biomedical research ethics board (BREB) of the University of Manitoba (Ethics File Number B2014:113). Written informed consent was obtained from all participants accepted into the study. The study was registered in the clinicaltrials.gov registry (Identifier: NCT02341924; www.clinicalTrials.gov).

Table 1. Summary of key nutrients and ingredients in the study food products