Plant sterol enriched functional food and atherosclerosis

Abstract

Hypercholesterolaemia is a major cardiovascular risk factor. A healthy diet and a healthy lifestyle reduces cardiovascular risk. ‘Functional foods’ supplemented with phytosterols are recommended for the management of hypercholesterolaemia and have become a widely used non‐prescription approach to lower plasma cholesterol levels. Two billion euros are spent world‐wide each year on various functional foods, which have regulator‐approved health claims for the management of elevated cholesterol levels. While international societies, such as the European Atherosclerosis Society or the National Heart Foundation in Australia, still advise phytosterols as an additional dietary option in the management of hypercholesterolaemia, recently released guidelines such as those from the National Institute of Health and Clinical Excellence in the United Kingdom are more critical of food supplementation with phytosterols and draw attention to significant safety issues. This review challenges whether an intervention with phytosterol supplements is beneficial. We summarize the current evidence from genetic diseases, genetic association studies, clinical trial data and data from animal studies.

Linked Articles

This article is part of a themed section on Principles of Pharmacological Research of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.11/issuetoc

Abbreviations

apoE

apolipoprotein E

CAD

coronary heart disease

EAS

European Atherosclerosis Society

NPC1L1

Niemann‐Pick C1‐like 1

Tables of Links

TARGETS
ABCG5
ABCG8
NPC1L1

LIGANDS
Ezetimibe
Laropiprant

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015).

‘Dosis sola facit venenum’

‘All things are poison and nothing is without poison; only the dose makes a thing not a poison’

Paracelsus (1493–1541)

Introduction

One in two humans in western societies dies as a result of cardiovascular disease (World Health Organization, 2014). Epidemiological studies show that cardiovascular diseases can be prevented and that lifestyle and diet play a key preventive role (Berry et al., 2012). There is evidence that regular physical activity and a balanced diet can reduce the risk of myocardial infarction (Lichtenstein et al., 2006). It is also known that individuals free of cardiovascular disease and cardiovascular risk factors increase their cardiovascular risk when they lead unhealthy lifestyles with poor dietary choices (King et al., 2011). Moreover, advertisements for nutritional supplements and so‐called functional foods promise apparent benefits to health. For this reason, millions regularly reach for these preparations. Evidence that these products are actually effective in reducing hard clinical end‐points, which is generally required for drugs, is not necessary, and there are doubts as to whether there are even any benefits to health by taking them (Weingärtner et al., 2009a; 2014a).

A western‐type diet contains about 200–500 mg cholesterol and up to 500 mg of phytosterols of which about 200–400 mg are plant sterols and only 50 mg are plant stanols (Weingärtner et al., 2009a). As natural constituents of plants, sterols are non‐nutritive compounds and differ from cholesterol only slightly in their structure by a methyl or an ethyl group at C‐24. Plant stanols are the saturated form of plant sterols; they have a double bond in the sterol ring. Saturation of plant sterols gives rise to plant stanols. Estimates for intestinal absorption range from 0.4 to 5% for plant sterols and from 0.02 to 0.3% for their saturated counterparts. As a consequence, plant stanol serum concentrations are about 10 times lower than plant sterol serum concentrations. Due to their lower absorbability and due to the fact that plant sterols are not synthesized in the human body, their concentrations are about 1000 times lower than cholesterol (Fransen et al., 2007; Weingärtner et al., 2009a; 2010; 2016). In 1951, sitosterol was first described as a therapeutic agent for hypercholesterolaemia (Peterson, 1951), and soon afterwards, Eli Lilly Company introduced ‘Cytellin’ as a pharmaceutical agent (Pollak and Krichevsky, 1981). However, due to its low water solubility and resulting low bioavailability, 18 g·day⁻¹ were needed to reduce serum cholesterol levels. Therefore, the product was unmarketable as a pharmacological agent and production was stopped soon after. In the beginning of the 90s, the Finnish chemist Ingmar Wester succeeded, by the esterification of plant stanols, in developing a process, which considerably improved the water solubility of phytosterols (Salo and Wester, 2005). The landmark study ‘Reduction of serum cholesterol with sitostanol‐ester margarine in a mildly hypercholesterolaemic population’ in 1995 in the New England Journal of Medicine set the pace for a rapidly growing market for so‐called functional foods in the prevention of cardiovascular disease (Miettinen et al., 1995). Of note, the Finnish researcher Ingmar Wester and the company RAISIO deliberately decided to saturate the naturally‐occurring plant sterols to form plant stanols before marketing their product, as plant sterols were suspected of having ‘pro‐atherogenic effects’ and the use of plant stanols reduces both serum levels of cholesterol and plant sterols (personal communication). This decision is even more remarkable as the saturation of plant sterols to plant stanols is an additional chemical step, which made the process more complex and the product more expensive.

High cholesterol levels are a known risk factor for cardiovascular diseases (Baigent et al., 2005). Many renowned international societies such as the European Atherosclerosis Society (EAS) recommend the use of phytosterols (plant sterols and plant stanols) as a food additive to reduce cholesterol levels (Gylling et al., 2014). In contrast, the German Drug Commission (Arzneimittelkommission der Deutschen Ärzteschaft, 2004) and the updated guidelines from the National Institute of Health and Clinical Excellence (NICE) in the United Kingdom advise against the general recommendation that plant sterols and plant stanols should be taken as a dietary supplement (National Institute for Health and Clinical Excellence, 2008), as there is a lack of endpoint data. The new EAS/European Cardiology Society guidelines also come to the conclusion that long‐term studies are needed to guarantee the safety of phytosterol‐enriched products (Rainer et al., 2011). The background for the currently controversial scientific debate is the phytosterols themselves (Patel and Thompson, 2006a,b; Weingärtner et al., 2009a,b). In clinical trials, plant sterols can indeed produce a desirable reduction in LDL cholesterol levels, by as much as 15% in hypercholesterolaemic subjects (Weststrate and Meijer, 1998). However, new studies show that this effect is not always reproduced with certainty and that phytosterol supplementation in some individuals can also lead to a paradoxical increase in cholesterol levels (Rideout et al., 2010; Weingärtner et al., 2016). Moreover, the reduction in cholesterol depends not only on the amount of the phytosterols taken but also on genetic differences in sterol metabolism (Vanhanen et al., 1993). Since patients with apolipoprotein E (APOE)4 homozygosity are ‘hyperabsorbers’ of cholesterol, they show a clearer reduction in cholesterol levels under phytosterol (and phytostanol) supplementation. The significance of elevated serum phytosterol (especially plant sterols) levels is unclear; this topic has been highly controversial for many years since some studies indicate its association with an increased cardiovascular risk, whereas others do not (Patel and Thompson, 2006a,b; Weingärtner et al., 2009a; Genser et al., 2012; Silbernagel et al., 2013a,b; Verges and Fumero, 2015). The purpose of this review is to summarize the current evidence on plant sterols and atherogenicity derived from patients with genetic disorders, genetic association studies, human studies and animal models.

The evidence from genetics

The discovery of sitosterolaemia, a rare inherited disorder, has raised curiosity as to whether plant sterols could be a possible risk factor for cardiovascular disease (Sudhop and von Bergmann, 2004). The classical description of sitosterolaemia in two sisters not only led to the concept that there was a mechanism preventing the accumulation of dietary plant sterols but also importantly suggested that a single gene may be responsible (Bhattacharyya et al., 1972). Although this disease is very rare, the investigation of two cases of fatal atherosclerotic events in boys under 21 years of age led to the identification of two other families and further strengthened the genetic component (Salen et al., 1985). A study of different families from all over the world led to the next breakthrough, the mapping of the sitosterolaemia locus, STSL, to human chromosome 2p21 (Berger et al., 1998; Patel et al., 1998a,b; Lee et al., 2001a) and shortly thereafter the gene defects were identified (Berge et al., 2000; Lee et al., 2001b). It is now established that two genes comprise the STSL locus, ABCG5 and ABCG8. The two gene products ABCG5 and ABCG8 are obligate heterodimers and function at the apical surface of hepatocytes and enterocytes to extrude sterols into the lumen and are the major cholesterol pumps, with a predilection to excrete non‐cholesterol sterols (Patel and Salen, 2010). These pumps perform a very basic physiological process in maintaining whole‐body sterol balance (Hazard and Patel, 2007). Failure of these pumps leads to elevated serum plant sterol levels. The plant sterol concentrations of patients with homozygous sitosterolaemia are elevated by up to 20‐fold, and these patients have xanthomas and suffer from premature onset of atherosclerosis, which is often fatal (Beaty et al., 1986). The fact that patients with this disease often experience aggressive progression of atherosclerosis despite practically normal cholesterol levels has led to the speculation that plant sterols themselves may have the potential to be atherogenic. Of note, comparable with patients with familial hypercholesterolaemia, not all patients with sitosterolemia develop relevant coronary artery diseases (Hansel et al., 2014). Mendelian randomization studies are particularly well suited to investigate the causality of biomarkers with disease phenotypes (Jansen et al., 2014). Using genome‐wide association and subsequent replication from the general population, we have identified a total of three genetic variants in ABCG8 and AB0, with significant effects on plasma plant sterol concentrations (Teupser et al., 2010). The same three variants were subsequently tested for an association with coronary artery disease in over 27 000 subjects. Strikingly, variants associated with a twofold‐increase in plasma plant sterols were associated with increased coronary artery disease, while variants associated with a decrease in plasma plant sterols were associated with decreased coronary artery disease. These findings are of importance, since the observations from sitosterolaemic patients are extended to the normal population of non‐sitosterolaemic humans and suggest that in these subjects, genetically determined twofold life‐long elevations in plasma plant sterols might lead to an increased risk to develop coronary artery disease (Teupser et al., 2010). This finding is of particular interest, since a diet supplementation with plant sterols leads to similar increases in serum plant sterol levels (O'Neill et al., 2005; Ras et al., 2013; Ras et al., 2016). However, plasma plant sterol concentrations are not only regulated by ABCG5, ABCG8 and ABO but also by the Niemann‐Pick C1‐like 1 (NPC1L1) protein (Altmann et al., 2004) (Figure 1). This protein is located in the apical membrane of the enterocyte and is the major cholesterol pump, with a predilection to absorb sterols (cholesterol and non‐cholesterol sterols) from the intestine (Klett and Patel, 2003). Recently, the Myocardial Infarction Genetics Consortium Investigators evaluated the association between inactivating mutations of the gene NPC1L1, serum cholesterol levels and the risk of developing cardiovascular disease in the normal population (Myocardial Infarction Genetics Consortium Investigators, 2014). Heterozygous carriers of NPC1L1 inactivating mutations had a mean LDL‐C level that was 120 mg·L⁻¹ lower than in non‐carriers, but resulted in a dramatic relative risk reduction of more than 50%. However, previous genetic analysis showed that a much greater life‐long reduction of LDL‐cholesterol by ~400 mg·L⁻¹ (1 mmol·L⁻¹) leads to a similar relative risk reduction of 54% (Ference et al., 2012) and data from the CTT analysis revealed that statin treatment results in a cardiovascular risk reduction by only 22% for each 1 mmol·L⁻¹ LDL‐cholesterol reduction (Baigent et al., 2005). These risk calculations are in accord with the hypothesis that the dramatic reduction in cardiovascular risk by inactivating mutations of NPC1L1 might not be explained by a 10% reduction in serum cholesterol levels alone, but potentially by an additional decrease in the absorption of ‘atherogenic’ non‐cholesterol sterols (plant sterols). In a similar manner, the effects of common genetic variants identified in our population‐based study (Teupser et al., 2010) had only minor effects on plasma cholesterol (−2, +0.9 and +1.5%), translating to absolute effects of −40, +20 and +30 mg·L⁻¹ for ABCG8 rs41360247, ABCG8 rs4245791 and ABO rs657152 respectively. As we know from these SNPs today, changes in total cholesterol are due to changes in LDL‐cholesterol. However, the effects of the three variants on coronary disease were −19, +10 and +9% and (in a similar matter as that for NPC1L1) much larger than would be expected from the minor changes in cholesterol. Taken together, these data further add to the hypothesis that plant sterols also increase cardiovascular risk in non‐sitosterolaemic humans (Weingärtner et al., 2015).

Figure 1.

Cholesterol and plant sterols are taken up in the enterocyte via NPC1L1. In the enterocyte, cholesterol and plant sterols are differentiated: most cholesterol molecules are esterified and are packed in chylomicrons. Up to 95% of plant sterols are extruded in the intestine via the obligate heterodimers ABCG5/ABCG8. Regulation of the expression of ABCG5/ABCG8 and NPC1L1 affects serum cholesterol and serum plant sterol levels and determines cardiovascular risk. MTP, microsomal transfer protein; ACTA: acyl‐CoA‐cholesterol‐acyltransferase.

The evidence from clinical studies

It has been demonstrated earlier that patients who consume food items (e.g. margarine) enriched with plant sterol esters have increased plant sterol concentration in their serum and aortic valve tissue (Weingärtner et al., 2008). Moreover, findings in that study demonstrated that patients who had consumed a diet supplemented with plant sterols over a time period of up to 4 years, when compared with those who had not, had sixfold higher concentrations of plant sterols in aortic valve tissue, indicating plant sterol deposits in cardiovascular tissue. These findings, however, could not be verified in a smaller study with a shorter period of plant sterol diet supplementation (Simonen et al., 2015). Luister et al. (2015) in another recently published study demonstrated a correlation between oxidized plant sterols in serum and deposits in aortic valve tissue. Earlier, Miettinen et al. (2005) and Helske et al. (2008) demonstrated a correlation between plant sterols in serum and in atherosclerotic lesions. However, the meaning of these correlations and possible deposits of plant sterols in cardiovascular tissue and whether they are indeed causally involved in the development of atherosclerosis has remained a matter of debate over the past decade (Patel and Thompson, 2006a,b; Weingärtner et al., 2009a; Genser et al., 2012; Silbernagel et al., 2013a,b; Verges and Fumero, 2015). Some case–control studies have demonstrated a positive association between plasma plant sterol concentrations and the risk of atherosclerotic cardiovascular disease (Glueck et al., 1991; Rajaratnam et al., 2000; Assmann et al., 2006; Weingärtner et al., 2009a,b; Matthan et al., 2009; Silbernagel et al., 2010; Weingärtner et al., 2011), and others did not confirm this association (Wilund et al., 2004; Pinedo et al., 2007). Three publications have even reported their association with decreased cardiovascular risk (Fassbender et al., 2008; Escurriol et al., 2010; Weingärtner et al., 2010). Moreover, a meta‐analysis showed no association between serum plant sterol levels and cardiovascular risk (Genser et al., 2012). However, the field is complicated by the lack of standardized measurements of plant sterols and, therefore, results from different laboratories are difficult to compare, and it is questionable if a meta‐analysis on this basis should be performed at all (Weingärtner et al., 2014a,b). The results of a recently published study from our group show an association between coronary artery disease and concentrations of campesterol oxides in aortic valve tissue (Luister et al., 2015). Moreover, we found a relationship between oxidized sitosterol : cholesterol ratio in the plasma and concomitant coronary heart disease (CAD). Contradictory results have also been reported with regards to the atherogenic effects of oxyphytosterols, particularly in animal studies, in in vitro experiments and in cytological investigations (Adcox et al., 2001; Maguire et al., 2003; Ryan et al., 2005; Abramsson‐Zetterberg et al., 2007; Vejux et al., 2012; Plat et al., 2014). Only limited data are available on the effects of an oxyphytosterol‐enriched diet and atherosclerosis. However, Plat et al. (2014) found that the consumption of a diet enriched with oxyphytosterols, similar to the consumption of oxysterols, increased atherosclerotic lesions in LDL^+/− mice. Also increased oxyphytosterol levels in patients with sitosterolaemia have led to speculations about potential pro‐atherogenic effects of plant oxidation products. The results demonstrated in our recently published study further support to this notion (Luister et al., 2015).

Previously, Silbernagel et al. (2013a) suggested that increased cholesterol absorption, but not increased plant sterol absorption, is associated with cardiovascular risk. They examined cholestanol as a marker of intestinal cholesterol absorption with respect to the association of six single nucleotide polymorphisms in ABCG8 and AB0 with cardiovascular risk. They reported that increased cholestanol : cholesterol ratios, but not the level of plant sterols in plasma, were associated with increased cardiovascular risk and concluded that increased absorption of cholesterol per se, rather than the absorption of plant sterols, was associated with atherosclerosis. However, in this analysis, multiple sterol measurement techniques were used, which is a major shortcoming of such an analysis (Weingärtner et al., 2014b). Moreover, Sudhop et al. (2002) found that patients with a positive family history of CAD exhibit significantly higher serum levels of the campesterol, sitosterol. However, no significant differences were found for cholestanol and the cholestanol : cholesterol ratio. Similarly, in our studies, there was no association between CAD and absolute values of cholestanol or its ratio to cholesterol in plasma and cardiovascular risk (Weingärtner et al., 2009b; Luister et al., 2015).

The evidence from animal studies

Ratnayake et al. (2000) reported that vegetable oils high in plant sterols reduce erythrocyte deformability and shorten the life span of rats. This experimental evidence was the cornerstone for the decision of Health Canada to ban plant sterol‐enriched products from the Canadian market shortly after their introduction in the year 2000 (Weingärtner et al., 2009a). With the introduction of the drug ezetimibe, the first pharmacological cholesterol absorption inhibitor, there was renewed interest in investigating the effects of blocking intestinal cholesterol absorption for therapeutic purposes (Meng, 2001). In a study by Weingärtner et al. (2008), the effect of inhibiting cholesterol absorption on atherosclerosis was investigated in Apoe ^−/− mice, using two different methods for the inhibition. The mice were fed either a high‐cholesterol western‐type diet with no supplement (control group), the same diet supplement with plant sterols, the same diet but with the addition of ezetimibe or a combination of these two options (ezetimibe and plant sterols) over a period of 6 months. It was found that inhibiting cholesterol absorption, whether by plant sterols, ezetimibe or a combination of both resulted in a dramatic reduction in cholesterol levels by as much as 8g·L⁻¹. In mice fed a low‐cholesterol diet, cholesterol levels were reduced from 4 to 2 g·L⁻¹. Each option used to reduce the cholesterol levels resulted in a significant reduction in the development of atherosclerotic lesions. The critical finding in this experiment, however, was that even though ezetimibe treatment and plant sterol diet supplementation resulted in comparable cholesterol levels, mice fed a plant sterol diet exhibited larger atherosclerotic lesions. Comparing mice with similar cholesterol levels, there was a strong correlation between plant sterol levels and atherosclerotic lesion size (r = 0.50), suggesting that plant sterols are directly associated with atherosclerosis. In two consecutive experiments, a diet supplementation with plant sterols was tested in wild‐type mice with regard to endothelial function and cerebral ischaemic lesions (Weingärtner et al., 2008). Wild‐type mice that were fed plant sterols had two fold higher serum phytosterols levels compared to controls, comparable cholesterol levels but exhibited impaired vasodilatation, which is an indicator of endothelial dysfunction. Moreover, mice on a plant sterol‐supplemented diet exhibited significantly increased ischaemic lesions after occlusion of the middle cerebral artery confirming the negative vascular effects of plant sterols in wild‐type mice (Weingärtner et al., 2008). However, the results of these experiments differ from those observed previously (Moghadasian et al., 1997; Wilund et al., 2004; Plat et al., 2006), probably because the effects of the plant sterol supplement were specifically tested at comparable serum cholesterol levels to control animals. In other words, these studies evaluated the net effect of increased plant sterol serum levels. Therefore, in both wild‐type mice and Apoe ^−/− mice, increased serum plant sterol levels at comparable serum cholesterol levels were associated with entirely negative vascular effects. Finally, Solca et al. (2013) and McDaniel et al. (2013) investigated the significance of plant sterol exclusion from the body in mice models. By feeding plant sterols to mice deficient in ABCG5/ABCG8 function, they found that plant sterols were toxic in mice unable to exclude plant sterols from their body. The accumulation of high levels of tissue plant sterol caused complex cardiac lesions, liver damage, hepatosplenomegaly and premature death in the animals as early as 5 weeks after starting a plant sterol‐supplemented diet. The authors concluded that the functionality of ABCG5/ABCG8 in excluding plant sterols from the organism is to protect the body from the toxic effects of plant sterols (McDaniel et al., 2013; Solca et al., 2013).

Against a backdrop of increasing evidence that plant sterols could be harmful as a food supplement, back in 2008, the German Federal Institute for Risk Assessment (Bundesinstitut für Risikobewertung) stated that these products should only be recommended for individuals with elevated cholesterol levels (Bundesinstitut für Risikobewertung, 2008). In December 2011, after additional results from clinical studies were published (Kelly et al., 2011), the German Federal Institute for Risk Assessment called for the general scrutiny of the use of phytosterols as a food additive at a European level and requested a reassessment by the European Food and Safety Authority (Bundesinstitut für Risikobewertung, 2011).

Due to the more relaxed approval and control conditions required for food additives compared to pharmacological preparations, investigations into the interactions of individual food additives, whether with each other or with drugs, have been mainly unsatisfactory. This is an important issue, especially for their consumption by elderly and infirm patients, children and individuals who are chronically ill with liver or kidney disease, high risk patients and patients taking a number of different drugs. Of note, for the pharmacological cholesterol absorption inhibitor ezetimibe, the US Food and Drug Administration have requested a hard cardiovascular outcome trial. Meanwhile, the IMPROVE‐IT trial has demonstrated the effectiveness and safety of inhibiting cholesterol absorption pharmacologically via inhibition of NPC1L1 and provided clear evidence that ‘hard cardiovascular outcomes’ are reduced (IMPROVE‐IT Investigators, 2015). For functional foods supplemented with plant sterols, such a trial is not even planned, because the expected beneficial effects on cardiovascular endpoints might be too small to be demonstrated in a feasible controlled trial (Silbernagel et al., 2015).

Conclusions

Positive lifestyle changes produced by incorporating physical activity and increasing the consumption of fresh fruits, vegetables and fish bring health benefits, a fact only recently demonstrated by the PREDIMED Study of the Mediterranean Diet. Clear evidence that functional foods supplemented with phytosterols are safe and effective in the prevention of cardiovascular diseases is as yet unavailable and individual studies show that they may even be harmful. For medicinal products, such questions must be clarified prior to authorization for market circulation, and it is only when the benefit outweighs the risk that a drug will actually be released onto the market. It is a fallacy to believe that any treatment leading to a reduction in LDL‐C levels leads to a parallel reduction in atherosclerosis. In the HPS2‐THRIVE trial, extended‐release niacin with laropiprant increased serious adverse events even though the reductions in LDL‐C were similar to those achieved in plant sterol studies (HPS2‐THRIVE Collaborative Group, 2014). However, due to the much slacker legislation, which governs dietary supplements and functional foods, there is no provision for this type of investigation into product safety, since ‘hard cardiovascular outcome studies’ are no prerequisite for the marketing of food supplements (Zawistowski and Jones, 2015). As long as results of trials testing relevant clinical endpoints for dietary supplements are pending, the general recommendation of these products will remain a matter of controversial debate.

Conflict of interest

OW received lecture fees and consulting fees from Merck. No other potential conflict of interest was reported relevant to this manuscript.

Köhler, J. , Teupser, D. , Elsässer, A. , and Weingärtner, O. (2017) Plant sterol enriched functional food and atherosclerosis. British Journal of Pharmacology, 174: 1281–1289. doi: 10.1111/bph.13764.