The Lipid-Lowering Effects and Associated Mechanisms of Dietary Phytosterol Supplementation

Abstract

Phytosterols (PS) are plant-based structural analogous of mammalian cholesterol that have been shown to lower blood cholesterol concentrations by ~10%, although inter-individual response to PS supplementation due to subject-specific metabolic and genetic factors is evident. Recent work further suggests that PS may act as effective triglyceride (TG)-lowering agents with maximal TG reductions observed in hypertriglyceridemic subjects. Although PS have been demonstrated to interfere with cholesterol and perhaps TG absorption within the intestine, they also have the capacity to modulate the expression of lipid regulatory genes through liver X receptor (LXR) activation. Identification of single-nucleotide polymorphisms (SNP) in key cholesterol and TG regulating genes, in particular adenosine triphosphate binding cassette G8 (ABCG8) and apolipoprotein E (apoE) have provided insight into the potential of utilizing genomic identifiers as an indicator of PS responsiveness. While PS supplementation is deemed safe, expanding research into the atherogenic potential of oxidized phytosterols (oxyphytosterols) has emerged with their identification in arterial lesions. This review will highlight the lipid-lowering utility and associated mechanisms of PS and discuss novel applications and future research priorities for PS pertaining to in utero PS exposure for long-term cardiovascular disease risk protection and combination therapies with lipid-lowering drugs.

Introduction

The impact of cardiovascular diseases (CVD) on American life cannot be over-emphasized as it presents major challenges to society through direct implications on public health, well-being, and economic prosperity. With about one third of American (approximately 800,000) deaths from CVD each year, CVD has become a primary health priority in the United States (U.S.) [1]. According to the Center for Disease Control and Prevention (CDC) analysis of data from the National Health and Nutrition Examination Survey (NHANES), nearly 50% of US adults over age 20 years (close to 107.3 million people) have at least one CVD risk factor [2], including obesity, hypertension, smoking, and high blood cholesterol [3]. As an established CVD risk factor, blood cholesterol levels are particularly troublesome as more than 100 million US adults have total blood cholesterol levels that far exceed the desirable target of ≤200 mg/dl [4]. Although first-line lifestyle approaches (diet and exercise) and drug therapy (statins, ezetimibe) have been largely effective in reducing low-density lipoprotein cholesterol (LDL-C) levels across the US population, close to 33% of US adults still have elevated LDL-C and there is concern that high-risk individuals often fail to meet their LDL-target goals.

Phytosterols (PS) are plant-based structural analogous of mammalian cholesterol with demonstrated cholesterol-lowering capability in both animal and human studies. As one of the most well-characterized dietary supplement approaches to lowering LDL-C concentrations, this review will provide an overview of the clinical utility of PS and discuss ongoing and emerging science related to PS metabolism.

PS Structure and Function

PS contain the same identical ring structure as cholesterol but differ in the side chain at the carbon 24 position of unsaturated double bonds and can differ in stereochemistry around chiral carbons [5, 6]. PS are classified as triterpene hydrocarbons and further distinguished based on the number of methyl groups on carbon 4. As a dietary component, PS are usually defined as a sterol containing a 28 or 29 carbon chain with methyl on carbon 4 which was removed (4-desmethyl sterol) [5]. Although plants contain a diverse number of PS, beta-sitosterol, campesterol, and stigmasterol are the most abundant in higher order plants and are most commonly consumed [5].

PS and their saturated form, phytostanols, are both integral parts of plants seeds and grains. Dietary sources typically include nuts such as almonds, cashews, coco seeds (unsweetened chocolate), and peanut butter [7]. In addition, oils and grains such as cereals are a major source of PS in the diet. Despite the abundant sources of PS, it is estimated that the western diet contains between 200 mg to 450 mg per day [8]. Although this amount is similar to cholesterol intake, it is markedly lower than the estimated 1,000 mg our ancestors obtained per day. In addition, this amount does not reach the recommended intake of 2 g/d that has been shown to result in any meaningful reduction in intestinal cholesterol absorption and blood cholesterol concentrations [7].

Cholesterol-Lowering Effects of PS

The total cholesterol (TC) and LDL-C lowering effect of PS supplementation is well characterized, backed by an extensive body of literature dating back to the early 1950s [9]. In response to the heightened importance of hypercholesterolemia in the formation and progression of atherosclerosis, the first PS-based drug, Cytellin, was introduced in 1954 [10]. Today, the advent of modern food science has facilitated the incorporation of PS into a variety of functional foods including low-fat yogurt, margarine, milk, vegetable oils [11] in addition to the formulation of capsule and tablet based supplements [12]. In the past, debate over the effectiveness of food-based versus capsule PS vehicles was highly contested, however recent clinical trials have demonstrated a similar LDL-C lowering response to oral PS supplements (capsules and tablets) versus fortified foods [13–17]. Current evidence suggests that ingestion of 2 g/d PS is generally associated with a 10% reduction of TC and LDL-C, however the absolute effectiveness of PS-mediated cholesterol lowering is affected by a variety of factors including dose, intake frequency, and individual baseline cholesterol concentrations [18–20]. Recent meta-analyses of clinical trials have demonstrated PS intake between 0.6–3.3 g/d, administered over the course of 4 to 52 weeks, results in a dose-response lowering of LDL-C concentrations by 6–14% [21, 22]. While the results demonstrate a dose-dependent cholesterol lowering response to stepwise increases of PS dosage (up to 3 g/d), variations in individual responses are apparent and will be discussed in detail later. Of note, while most PS feeding trials observed no change in high-density lipoprotein cholesterol (HDL-C), PS associated reductions of plasma LDL-C, small-dense LDL particles (LDL-p), and apolipoprotein (apo) B-100 may confer CVD protection in at-risk populations by improving the HDL/LDL and apoB-100/apoA-1 ratios [23–25]. However, beyond improvements in surrogate lipid risk markers, a hard endpoint prospective study to evaluate the effects of PS in protecting against CVD events has not been conducted.

TG Lowering Effects of PS

Due to the intensive focus on the cholesterol lowering action of PS as a means to attenuate CVD risk, the triglyceride (TG) lowering properties of PS were initially overlooked but have recently been the focus of increasing research. While the primary outcomes of recently published human and animal studies primarily focused on the lowering of TC and LDL-C, analyses of secondary outcomes have highlighted the capacity of PS to modulate plasma and hepatic TG concentrations [14, 17, 23, 26]. Further, pooled analyses conducted by Naumann et al. [27] and Demonty et al. [28] have exhibited a relationship between greater reductions in circulating TG concentrations in response to PS treatment and increased baseline TG levels. Increased PS intake from 2 to 4 g/d resulted in greater reductions of plasma TG as baseline TG levels rose from 1 to 3 mmol/L [27], whereas an intake of roughly 2 g/d PS resulted in a 6% reduction with greater reductions observed in subjects with a baseline TG level of at least 1.9 mmol/L [28]. A number of trials have been conducted to directly assess the TG lowering response to PS treatment. Theuwissen et al. [29] demonstrated the relationship between baseline TG concentrations and responsiveness to a fixed PS dosage. Subjects with baseline TG levels between 1.73–2.3 mmol/L experienced no reduction in circulating TG concentrations in response to 2.5 g/d PS, whereas an 11% reduction was observed in subjects with baseline TG levels ˃ 2.3 mmol/L. In hypertriglyceridemic metabolic syndrome subjects, Plat et al. (2009) reported a TG-lowering effect of 27.5% in response to consumption of a plant stanol (2g/d) yogurt drink for 9 weeks [30]. More recently, Blumenfeld Olivares et al. [31] reported a non-significant TG reduction (−5.88 mg/dL) in hypertriglyceridemic children supplemented with 2.24 g/d of PS.

In support of the clinical trial data, several pre-clinical animal studies have been conducted to specifically characterize the TG-lowering properties of PS. Diet-induced dyslipidemic Syrian golden hamsters fed a high fat (HF) diet supplemented with 2% PS resulted in a 49% reduction of blood TG compared to the non-PS HF group [32]. Interestingly, reductions in blood TG in the PS group were comparable to reductions seen in a joint branch of the study supplemented with ezetimibe, a novel and potent cholesterol-lowering drug agent with TG lowering effects [33]. Similar results were observed in a HF-1% PS diet supplemented hamster model, where the introduction of PS at day 15 of a HF diet regimen was able to reduce plasma and hepatic TG concentrations to those of the chow fed control group [34]. Although the lowering of TG in response to PS appears consistent amongst several diet-induced animal models, a certain amount of discord exists with regard to the degree of TG-lowering and the associated mechanisms, factors which can likely be attributed to the type of diet used, PS dosage, study duration, and diurnal regulation of lipid levels [35–38].

Responders/Non-Responders to Phytosterols

The results of the meta-analyses mentioned above highlight an apparent dose dependent response to PS, however the cholesterol lowering responses are often non-linear and possess a degree of inter-individual variability. This individual variability observed in controlled feeding studies has lead researchers to classify subjects as responders and non-responders to PS therapy [39]. Recent work has sought to characterize the phenotypic and genomic traits of individuals that may underscore the large range of lipid-lowering responses to PS intervention. In a retrospective study, Rideout et al. [40] demonstrated a correlation between high basal cholesterol synthesis rates and decreased LDL-C responsiveness to PS treatment compared to those with low basal cholesterol synthesis. Follow up work by Mackay et al. [41] utilizing the lathosterol-to-cholesterol ratio (L:C), a surrogate marker of cholesterol synthesis, in mildly hypercholesterolemic adults supports the hypothesis that cholesterol synthesis phenotype is predictive of PS responsiveness, with low cholesterol synthesizers having the greatest LDL-C lowering response to PS intervention. The use of single-nucleotide polymorphisms (SNP) as genetic markers of plasma PS and cholesterol concentrations has emerged as a promising avenue to characterize individual responsiveness to PS treatment. A genome-wide analysis of samples from the Cooperative Research in the Region of Augsburg (KORA) and CARdiovascular disease, Living and Ageing in Halle (CARLA) cohorts showed adenosine triphosphate binding cassette (ABC) G8 SNPs rs41360247 and rs4245791 were independently associated with plasma PS levels. In addition, ABCG8 rs41360247 was associated with a decreased risk of coronary artery disease (CAD), while ABCG8 rs4245791 and rs657152 were associated with increased CAD risk [42]. A meta-analysis of small cross-sectional cohorts found ABCG8 SNP rs6544718 (p.A632V) was associated with a 0.11 mmol/L reduction in plasma LDL-C concentrations when compared to those with the common 632A variant. Similarly, the ABCG8 polymorphism rs11887534 (p.D19H) was associated with decreased PS levels and by extension increased lathosterol concentrations, indicative of increased intestinal sterol loss [43]. Hornstein et al. [44] showed ABCG8 variant rs137852988 (G574R) was associated with a 35%−37% increase in PS levels and decreased lathosterol concentrations.

Additional investigation into apoE variants (E2, E3, E4) have been conducted, with mixed results. Following a 3 month intervention phase where 75 mildly-hypercholesteremic subjects were given a standard healthy diet, Bañuls et al. [45] supplemented 2 g/d PS to half the cohort for an addition 3 month period. As expected, the PS cohort experienced decreases in TC (5.1%) and LDL-C (8.1%) compared to the non-PS supplemented group, however these changes were independent from apoE genotype. Conversely, Mackay et al. [46] found positive associations between cholesterol 7 alpha-hydroxylase (CYP7A1) rs3808607 G/T and G/G as well as apoE ε3 and ε4 isoforms and decreased LDL-C, with apoE ε4 participants experiencing greater reductions in response to PS treatment. Further, subjects with the genoset CYP7A1 rs3808607 T/T/apoE ε3 were identified as non-responders to PS treatment which may prove to be a novel and useful genomic indicator to PS responsiveness. Additional polymorphisms related to cholesterol and TG metabolism have been studied, in particular cholesteryl ester transfer protein (CETP) rs4148217 and rs5882, however the varied responses will require further efforts to fully categorize their respective nutrigenomic potential [46, 47]. Collectively, the utility of SNPs as predictive markers of PS responsiveness remains uncertain but worthy of further investigation in the light of future personalized nutrition strategies.

Lipid-Lowering Mechanisms

PS-mediated regulation of plasma cholesterol levels is in large part facilitated through decreased cholesterol absorption and transport within the small intestine [Figure 1]. Under normal physiological conditions, nonesterified cholesterol is incorporated into bile salt micelles for presentation to the brush border of enterocytes where membrane bound transport proteins facilitates absorption [48]. However, in the presence of PS a decrease in cholesterol incorporation into micelles is observed. It is widely accepted that decreased cholesterol micellarization is due to the innate hydrophobic structural properties of PS which competes with and displaces cholesterol resulting in increased fecal loss [49]. The Niemann-Pick C1-like 1 (NPC1L1) transport protein is well known for facilitating unidirectional intestinal cholesterol absorption [50, 51]. Traditionally, PS were thought to compete with cholesterol for absorption into enterocytes via NPC1L1 therefore contributing to further cholesterol fecal loss [52], however, recent work by Zhang et al. demonstrates an inability of PS to bind the N-terminal domain of NPC1L1 which structurally contributes to high specificity of NPC1L1 for cholesterol transportation into the cytosol of enterocytes [53]. While NPC1L1 is generally believed to be the primary PS transporter within the intestine, further research is needed to detail the precise mechanisms by which PS and NPC1L1 may interact to facilitate a decrease in cholesterol absorption.

Figure 1:

Proposed cholesterol-lowering mechanisms of PS. Intestinal PS mediated lowering of cholesterol is facilitated through decreased micellarization of cholesterol esters resulting in decreased absorption and increased fecal sterol loss. Decreased intestinal cholesterol status activates the transcription factor SREBP2, resulting in the increase of lipoprotein receptors LDLr and SR-B1, facilitating the excretion of plasma cholesterol via the TICE pathway. Decreased expression of NPC1L1 and diminished activity levels of ACAT-2 and MTP coupled with the activation of LXR-mediated expression of ABCG5/G8 further inhibit cholesterol absorption and packaging into chylomicrons. Similarly, reduced hepatic cholesterol concentrations result in SREBP2 activation resulting in a subsequent upregulation of LDLr and HMG-CoAr. Hepatic LXR activation also induces cholesterol efflux transporters ABCG5/G8 and ABCA1 resulting in reduced hepatic cholesterol status.

Abbreviations: adenosine triphosphate binding cassette A1 (ABCA1), adenosine triphosphate binding cassette G5/G8 (ABCG5/G8), acyltransferase-2 (ACAT-2), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAr), liver X receptor (LXR), microsomal triglyceride transport protein (MTP), Niemann-Pick C1-like 1 (NPC1L1), phytosterols (PS), scavenger receptor class B type 1 (SR-B1), sterol response element binding protein 2 (SREBP2), transintestinal cholesterol excretion (TICE)

Once absorbed, intracellular intestinal regulation of cholesterol homeostasis occurs through the activity of acyltransferase-2 (ACAT-2) and microsomal triglyceride transport protein (MTP), responsible for the esterification of free cholesterol and subsequent packaging of cholesterol esters into nascent chylomicrons, respectively [54, 55]. In the presence of PS, the activity levels of ACAT-2 and MTP have been shown to be decreased, thereby decreasing the secretion of cholesterol and TG into circulation [56, 57]. PS and their metabolites have also been identified as potent liver X receptor (LXR) ligands and as a result have significant effects on downstream target genes involved in cholesterol absorption, packaging, and efflux [58, 59]. Within intestinal enterocytes, activation of LXR has been to shown to upregulate the expression of ABCG5/G8, membrane bound heterodimer transport proteins responsible for the efflux of PS and cholesterol from the cytosol of enterocytes into the lumen of the small intestine for excretion [60, 61].

Differential mRNA expression of NPC1L1 has also been observed in response to PS ingestion, decreased in apoE −/− mice while increased in C57BL/6J mice, likely secondary to varied degrees of cholesterol absorption inhibition within the small intestine [62]. The combined downstream effects of PS on intestinal LXR target genes including ABCG5/G8 and NPC1L1, as well as reduced ACAT-2 and MTP activity offer a synergistic mechanism for the observed decrease of plasma cholesterol as well as the lower relative physiological concentration of blood PS compared to cholesterol. Interestingly, PS were shown to attenuate the LXR mediated expression of the basolateral sterol efflux protein ABCA1 by inhibiting the synthesis of the oxysterol 27-hydroxycholesterol (27HC), a known LXR agonist, in Caco-2 enterocytes [63]. ABCA1 controls the basolateral efflux of intracellular cholesterol onto nascent HDL and is therefore critical for effective reverse cholesterol transport in peripheral tissues [64]. In this study, the addition of sitosterol or campesterol and cholesterol (PS+C) in vivo inhibited the conversion of intracellular cholesterol to 27HC by reducing the expression of the sterol 27-hydroxylase (CYP27) [63]. The impact of PS modifications of oxysterol synthesis is unknown, however, due to the atherogenic nature of plasma and tissue oxysterol concentrations [65], this metabolic process may offer further insight into the cardioprotecive capacity of PS.

Sterol response element binding protein 2 (SREBP2) is a key regulator of cholesterol homeostasis. In response to reduced intracellular cholesterol concentrations, inactive cytosolic SREBP2 embedded in the endoplasmic reticulum undergo proteolytic cleavage and enter the nucleus acting on the promoter regions of key cholesterol regulating proteins [66]. In theory, initiation of hepatic SREBP2 activation should occur secondary to the hypocholesterolemic effects of PS in the small intestine, however PS have been shown to inhibit this process in both mouse and hamster models [61, 67]. Interestingly, intestinal SREBP2 activity was increased in response to PS gavage resulting in a five and six-fold increase of intestinal LDL receptor (LDLr) and proprotein convertase subtilisin/kexin type 9 (PCSK9) expression, respectively [67]. As noted by the authors, such dramatic increases of intestinal lipid transport gene expression may be indicative of increased transintestinal cholesterol excretion (TICE). TICE represents an alternative pathway for cholesterol removal, independent of hepatobiliary pathways, by which circulating cholesterol is reabsorbed by enterocytes and excreted into the intestinal lumen. While the exact mechanisms are unclear, expression of genes related to basolateral lipid transportation, LDLr, PCSK9, and scavenger receptor class B type 1 (SR-B1) have been identified as active components for sterol influx, while ABCA1, ABCG5/G8, and possibly NPC1L1 expression may be coupled to facilitate cholesterol efflux into the lumen [68, 69]. Due to potential PS mediated SREBP2 and LXR activation, the role of PS modulation of TICE is an interesting prospect and requires further work.

The exact pathway(s) by which PS reduce TG concentrations is not yet precisely known, however, there is evidence to suggest that alterations in intestinal TG and/or fatty acid (FA) metabolism is at least partly responsible for the PS-induced TG-lowering response [Figure 2]. Liang et al. [56] reported a blood TG reduction of 28% in male Syrian hamsters fed diets supplemented with β-sitosterol or stigmasterol which was related to decreased mRNA expression of intestinal MTP, which plays a vital role in the secretion of apolipoprotein B (apoB)-containing chylomicron particles from the intestine. Finally, previous preclinical studies have also demonstrated that increased fecal FA excretion may underlie the TG lowering response to phytosterols [34] [36].

Figure 2:

Proposed triglyceride-lowering mechanism of PS. Reductions in hepatic and plasma TG concentrations in response to PS may be attributed to the physical interference of intestinal absorption and diminished incorporation into chylomicrons by the down regulation of MTP, resulting in increased fecal fat loss. PS have also been shown to reduce the expression of FAS, a key regulator of de novo lipogenesis, in addition to reducing hepatic VLDL secretion and particle number.

Abbreviations: fatty acid synthase (FAS), fatty acid translocase/CD36 (FAT/CD36), fatty acid transport protein (FATP), microsomal triglyceride transport protein (MTP), phytosterols (PS), triglycerides (TG), very low-density lipoprotein (VLDL)

Aside from potentially altering intestinal FA absorption, PS have also been suggested to alter hepatic fat metabolism, a central site of FA and TG metabolism including de novo lipogenesis and the synthesis and uptake of TG-rich containing lipoproteins. PS consumption has been shown to reduce large and medium plasma very-low density lipoprotein (VLDL) particles in human dyslipidemic metabolic syndrome subjects [70]. This reduction in VLDL particles appears to be related to alternations in hepatic VLDL secretion as Plat et al., recently reported a reduction in hepatic VLDL secretion in male C57BL/6J mice fed a high fat diet supplemented with 3.1% PS or stanol esters for 3 weeks [35]. Additionally, we recently reported a reduction in de novo lipogenesis and an associated down-regulation of hepatic fatty acid synthase (FAS) protein abundance in adult male Syrian golden hamsters fed a high fat diet supplemented with PS [32].

Safety

The US Food and Drug Administration, the European Food Safety Authority, and Health Canada have approved a health claim for plant sterol-fortified food products in the reduction of coronary heart disease risk [71]. Although there is still some debate about the safety of PS with respect to atherosclerosis (see below), the overwhelming majority of experts believe there to be no toxicity at recommended intake levels. A 9-part evaluation on the safety of PS with respect to oestrogenicity, toxicology, reproductive performance, offspring growth and development, gut bacterial profile, mutagenic activity, genotoxicity of PS oxides, and adverse effects, had been conducted as an extensive measure to test PS toxicity in a combination of in vitro, rat, and human trials. In all parts of the study, there were no abnormal findings that would suggest a harmful effect of PS [72–80].

Supraphysiological concentrations of PS, as seen in sitosterolemic individuals, is considered a safety concern for this sub-population. Due their genetic abnormalities, these patients have extremely high levels of circulating PS that is ultimately associated with early onset of CVD [81]. However, the concentration of PS found in sitosterolemia patients are orders of magnitude higher than that observed in normal individuals from regular diet or from supplementation [81]. Weingärtner et al. reported that PS supplementation was associated with endothelial dysfunction and increased deposition in the brain and liver of apoE −/− mice [82]. Similarly, Glueck et al. demonstrated that PS were a risk factor for coronary artery disease independent of cholesterol, an observation supported by Miettinen et al. who demonstrated the presence of PS in atheromatous plaques [83, 84]. However, the effects of PS as a potential risk factor in CVD is speculative and needs further research. Much of the discrepancy related to the pro or anti-atherogenic effects of PS may be related to oxyphytosterols, oxidized derivatives of PS.

Increased presence of the oxidative products of cholesterol (oxysterols) and PS (oxyphytosterols) in vascular tissues have generated concerns of safety and subsequent roles in early CVD development [85, 86]. As discussed above, the question of PS safety remains highly contested due in part to the lack of substantial evidence of CVD risk associated with plasma PS levels and the presence of PS and their oxidative products within aortic valves and arterial lesions in mice and humans [87–90]. Additionally, elevated oxyphytosterol levels in plasma and arterial walls have been shown to be associated with elevated reactive oxygen species (ROS) within the aorta of apoE −/− mice, however no detrimental impact on vascular function was observed [91]. Unlike the aforementioned studies, which utilized observational data and pre-clinical oxyphytosterol feeding studies to assess CVD risk, recent studies have begun to assess the effects of PS enriched foods on the development of oxyphytosterols in controlled feeding trails.

A series of studies conducted by Baumgartner et al. [92, 93] demonstrated varying responses to PS supplementation on serum oxyphytosterol concentrations in fasted and postprandial states. Subjects were fed a PS, stanol, or control margarine for three intervention periods lasting 4 week each, separated by 4 week washout periods. Supplementation of PS- fortified margarine, equivalent to 3 g/d, showed no increase in fasting plasma oxyphytosterol concentrations whereas plant stanol consumption decreased the concentrations of 7β-OH-campesterol and 7-keto-campesterol [92]. A subset from this cohort was selected for a follow on study by which after completing the final 4-week intervention period, were fed a PS, stanol, or control mixed meal shake followed by a non-cholesterol or PS contained mixed-meal shake 4 hours later. Plasma oxyphytosterol concentrations were taken at baseline and throughout the wait times after ingestion of the first and second meals, the intervention was repeated utilizing the previously described methods [93]. Postprandial 7β-OH-campesterol and 7β-OH-sitosterol concentrations were elevated in the PS mixed meal shake groups, however this effect was only observed after the ingestion of the second shake suggesting the role of enterohepatic recycling of oxidative PS that are excreted in the lumen of small intestine and reabsorbed as oxyphytosterols [93]. While intriguing to speculate the exact mechanisms surrounding the time delay of oxyphytosterol concentrations following ingestion, the exact mechanisms and implications remain unknown. Possible explanations for the discord between specific oxyphytosterol concentrations may be related to individual factors influencing oxyphytosterol production [94]. In addition, preferential oxidation of specific PS and subsequent efflux from peripheral tissues into the blood stream for excretion may lend insight into the apparent time-dependent changes in concentrations observed in feeding studies [95].

Novel Applications and Future Research

Recent work from our lab has focused on the use of PS supplementation as a novel treatment option for attenuating excessive maternal gestational hypercholesterolemia with an overall aim to reduce offspring CVD risk by limiting excessive fetal cholesterol exposure in utero. Fetal exposure to excessive fat and cholesterol as a result of maternal diet-induced or genetic dyslipidemia during pregnancy has been shown to increase fetal plasma cholesterol concentrations and aortic fatty streak formation and predispose adult offspring to diet-induced obesity, hyperlipidemia, and arterial plaque development [96, 97]. Female apoE −/− mice were fed a cholesterol-rich (CH) (0.15%) diet or cholesterol-rich diet supplemented with 2% PS (CH/PS) throughout gestation and lactation at which time serum cholesterol, lipoproteins, and TG levels were assessed in both dams and pups [26]. Compared to the CH fed groups, CH/PS dams demonstrated decreased serum and hepatic cholesterol (−55%, −69%), LDL-C (−47%), and non-HDL cholesterol (−56%). Similarly, pups from CH/PS fed mothers displayed decreased serum and hepatic cholesterol (−25%, −38%), LDL-C (−47%), and TG (−41%) [26]. Similar results were observed in female Syrian golden hamsters [98] fed Chow (Chow), chow with 0.5% (CH) diet, or CH diet with 2% PS (CH/PS) through gestation and lactation. Compared to pups from dams fed Chow, pups from CH dams showed increased levels of TC (+68%), LDL-C (+154%), HDL-C (+30%), and non-HDL-C (+123%). Maternal PS supplementation was able to reverse and lower TC (−26%), non-HDL-C (−32%), LDL-C (−29%), as well as normalize mRNA expression of LDLr and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) levels to those of pups from chow fed mothers [98]. The observed alterations of lipid status in both apoE −/− and hamster models were independent of feed intake and the trajectory of body weight gain in dams during the gestation and lactation periods. Similarly, litter weights during the postnatal period and the male-to-female sex ratio did not differ (p>0.05) between litters in each respective study [26, 98]. These results suggest a potential role for PS in protecting against the maladaptive programming responses associated with excessive cholesterol exposure. However, it still has yet to be demonstrated that these early improvements in lipid profile will translate into adulthood.

Another novel application and potential area of growth for PS may be as effective adjuncts to drug-based lipid-lowering strategies. Due to the capacity of PS supplementation to reduce CVD risk factors, combination based therapies utilizing statins are an interesting option for investigating potential additive effects of pharmaceutical/nutraceutical interventions. De Jong et al. [99, 100] assessed the effects of PS supplementation in conjunction with statin use on lipid metabolism, inflammation, and endothelial function. An additive LDL-C lowering effect of roughly 10% was seen with PS supplementation when compared to non-PS supplemented statin users, however, no change in markers of inflammation or endothelial function was observed. A follow up study from the same cohort found a positive correlation between increased serum campesterol concentration and retinal vasculature diameter [101].

Ezetimibe (EZ) is considered the most effective drug approach in reducing intestinal cholesterol absorption which functions by directly targeting NPC1L1, the intestinal cholesterol transport protein [102]. The cholesterol lowering response to combined PS and EZ therapy is controversial, as previous pre-clinical [103] and clinical investigations [104, 105] have reported both positive and negative findings. In the only other animal experiment, Batta et al. [103] observed a paradoxical increase in plasma cholesterol in response to a PS and EZ derivative (SCH 58235) combination in wild-type Kyoto (WKY) rats fed a standard rodent chow diet. However, as WKY rats are a sitosterolemic model characterized by hyperabsorption and reduced elimination of dietary PS [106], the clinical implications of this study for the general population are unclear.

To date, two clinical studies have been conducted to examine the cholesterol-lowering potential of a PS/EZ combination. Although Jakulj et al. [104] reported no therapeutic benefit of a PS/EZ combination (2 g PS/d, 10 mg/d EZ) over either monotherapy in forty mildly hypercholesterolemic subjects, a recent study by Lin et al. [105] reported a further 7% reduction in LDL-C following a PS/EZ combination compared with EZ treatment alone in a similar study population of twenty-one subjects. The discrepancies in these clinical study outcomes are difficult to reconcile but may be attributable to study design factors including feeding protocol (free-living vs controlled diet), study length (4 vs 3 weeks), plant sterol dose and vehicle (2 g in spread vs 2.5 g in soybean oil), and timing of PS dose (not controlled vs morning and evening).

Summary

With a long history of research characterizing the cholesterol-lowering effects and underlying mechanisms of PS, these plant-based bioactive components should be emphasized as an important component of preventative health care strategies to reduce LDL-C concentrations. Moreover, the more recent characterization of PS as TG-lowering agents, particularly in hypertriglyceridemic individuals, may expand the clinical utility of PS. Future research priorities for PS may be found in novel applications including pharmaceutical/nutraceutical combination lipid-lowering approaches. To fully advance the clinical application of PS, knowledge gaps in our understanding of inter-individual lipid-lowering responsiveness and potential risks associated with oxidized phytosterols will require more attention.