**Interactions of Oxysterols with Atherosclerosis Biomarkers in Subjects with Moderate Hypercholesterolemia and Effects of a Nutraceutical Combination (***Bifidobacterium longum* **BB536, Red Yeast Rice Extract) (Randomized, Double-Blind, Placebo-Controlled Study)**

**Stefania Cicolari 1, Chiara Pavanello 1,2, Elena Olmastroni 3, Marina Del Puppo 4, Marco Bertolotti 5, Giuliana Mombelli 6, Alberico L. Catapano 1,7, Laura Calabresi 1,2 and Paolo Magni 1,7,\***


**Abstract:** Background: Oxysterol relationship with cardiovascular (CV) risk factors is poorly explored, especially in moderately hypercholesterolaemic subjects. Moreover, the impact of nutraceuticals controlling hypercholesterolaemia on plasma levels of 24-, 25- and 27-hydroxycholesterol (24-OHC, 25-OHC, 27-OHC) is unknown. Methods: Subjects (*n* = 33; 18–70 years) with moderate hypercholesterolaemia (low-density lipoprotein cholesterol (LDL-C:): 130–200 mg/dL), in primary CV prevention as well as low CV risk were studied cross-sectionally. Moreover, they were evaluated after treatment with a nutraceutical combination (*Bifidobacterium longum* BB536, red yeast rice extract (10 mg/dose monacolin K)), following a double-blind, randomized, placebo-controlled design. We evaluated 24-OHC, 25-OHC and 27-OHC levels by gas chromatography/mass spectrometry analysis. Results: 24-OHC and 25-OHC were significantly correlated, 24-OHC was correlated with apoB. 27-OHC and 27-OHC/total cholesterol (TC) were higher in men (median 209 ng/mL and 77 ng/mg, respectively) vs. women (median 168 ng/mL and 56 ng/mg, respectively); 27-OHC/TC was significantly correlated with abdominal circumference, visceral fat and, negatively, with high-density lipoprotein cholesterol (HDL-C). Triglycerides were significantly correlated with 24-OHC, 25-OHC and 27-OHC and with 24-OHC/TC and 25-OHC/TC. After intervention, 27-OHC levels were significantly reduced by 10.4% in the nutraceutical group Levels of 24-OHC, 24-OHC/TC, 25-OHC, 25-OHC/TC and 27-OHC/TC were unchanged. Conclusions: In this study, conducted in moderate hypercholesterolemic subjects, we observed novel relationships between 24-OHC, 25-OHC and 27-OHC and CV risk biomarkers. In addition, no adverse changes of OHC levels upon nutraceutical treatment were found.

**Keywords:** oxysterols; 24-OHC; 25-OHC; 27-OHC; cholesterol metabolism; probiotic; cardiovascular risk; hypercholesterolemia; monacolin K; nutraceutical

**Citation:** Cicolari, S.; Pavanello, C.; Olmastroni, E.; Puppo, M.D.; Bertolotti, M.; Mombelli, G.; Catapano, A.L; Calabresi, L.; Magni, P. Interactions of Oxysterols with Atherosclerosis Biomarkers in Subjects with Moderate Hypercholesterolemia and Effects of a Nutraceutical Combination (*Bifidobacterium longum* BB536, Red Yeast Rice Extract) (Randomized, Double-Blind, Placebo-Controlled Study). *Nutrients* **2021**, *13*, 427. https://doi.org/10.3390/nu13020427

Academic Editor: Roberto Cangemi Received: 21 December 2020 Accepted: 26 January 2021 Published: 28 January 2021

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#### **1. Introduction**

Several lipid biomarkers may contribute to atherosclerosis-related cardiovascular diseases (ASCVD) and, among them, low-density lipoprotein (LDL) is a well-established causal factor [1]. Cholesterol metabolism includes de-esterification in lysosomes to generate free cholesterol which is used for several cellular processes [2] and could also undergo enzymatic oxidation in the mitochondria, leading to the formation of oxysterols [3]. These cholesterol metabolites, precursors of bile acids, are involved both in physiological mechanisms, interdependent with lipid and glucose metabolism, as well as in biological functions such as immune and cerebral homeostasis [4,5]. Additionally, oxysterols have been related to some pathological processes (e.g., atherosclerosis, type 2 diabetes mellitus, neurodegenerative disorders, cancer), for which they may represent potential innovative biomarkers [6]. We focused our attention on 24-hydroxycholesterol (24-OHC), 25-hydroxycholesterol (25- OHC) and 27-hydroxycholesterol (27-OHC), which are mainly synthesized by cytochrome P450 family 46 subfamily A member 1 (CYP46A1), cholesterol 25-hydroxylase (CH25H) and cytochrome P450 family 27 subfamily A member 1 (CYP27A1), respectively. Interestingly, these oxysterols have been shown to act as a link between cholesterol metabolism and different physiological systems [4,7].

Moderate hypercholesterolemia is frequently observed in subjects with medium/low 10 years CV risk, represents a significant population burden, particularly when combined with unhealthy lifestyle habits [8] and is often underdiagnosed and undertreated, therefore highly contributing significantly to ASCVD prevalence [9]. Few studies have evaluated the circulating levels of 24-, 25- and 27-OHC in subjects with moderate hypercholesterolemia, also after statin treatment [10,11].

Therapeutical strategies for this condition may include the use of low-efficacy/lowdose statins and/or nutraceutics [12–14]. This treatment may offer multi-faceted effects and significant advantages over no-treatment or inadequate adherence to drug treatment, sometimes due to adverse effects and other reasons [15–17]. To date, information is lacking about the impact of nutraceuticals, targeted to improve the atherogenic lipid profile, on the synthesis of oxysterols, downstream of inhibition of cholesterol biosynthesis and absorption. Based on these considerations, the main objective of the present study was to evaluate the interactions of oxysterols with cardiovascular biomarkers and subsequently to study the effects of a nutraceutical treatment (*Bifidobacterium longum* BB536, RYR extract, niacin, coenzyme Q10) on their circulating levels. This nutraceutical combination was previously found to be quite effective in reducing LDL cholesterol (LDL-C) and total cholesterol (TC) levels in moderately hypercholesterolemic subjects [18]. The study yielded novel data on these biochemical events, with potential health implications and supported the safety profile of this nutraceutical combination.

#### **2. Materials and Methods**

#### *2.1. Study Design and Population*

The study was conducted at the Centro Dislipidemie (ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy) in the period 2015–2017, according to the guidelines of the Declaration of Helsinki. The study cohort included 33 subjects (16 males and 17 females) with moderate hypercholesterolemia, median age: 57 years (Q1 = 48 and Q3 = 63 years) and low total CVD risk at (0%: 8 subjects; 1%: 15; 2%: 5; 3%: 3; 4%: 1; 5%: 1), assessed by the SCORE Risk Charts for low risk countries (like Italy) [19]. Inclusion criteria were subjects in primary CV prevention, age: 18–70 years, non-smokers, LDL-C: 130–200 mg/dL. Exclusion criteria included: pregnancy, smoking (current or previous), diagnosis of diabetes mellitus, chronic liver disease, renal disease, or severe renal impairment treated with insulin or antidiabetic drugs; untreated, uncontrolled or severe arterial hypertension; obesity (body mass index (BMI) >30 kg/m2); any pharmacological treatments known to interfere with the study treatment (including statins, ezetimibe, fibrates, thyroid hormones); and patients enrolled in another research study in the past 3 months. The study cohort included

5/32 subjects (15.6%) undergoing drug therapy for arterial hypertension, as reported in [18], together with average food intake according to sex.

The same cohort was also included in a 12-week intervention study (a randomized controlled trial (RCT) design with parallel-groups (NCT02689934)). Subjects were randomly assigned to receive either placebo (1 sachet/d; *n* = 17) or a nutraceutical combination (Lactoflorene Colesterolo®-1 sachet/d; granules for oral suspension; with taste/appearance identical to the placebo sachet) composed of 1 bn UFC *Bifidobacterium longum* BB536, RYR extract (10 mg monacolin K), 16 mg niacin, 20 mg coenzyme Q10; *n* = 16) or (Figure 1; CONSORT flow diagram). The randomization table was produced by computer-generated random numbers. The prevalence of subjects with drug-controlled hypertension was 18.8% in the placebo arm and 12.5% in the intervention arm. The study was approved by the Ethics Committee of ASST Grande Ospedale Metropolitano Niguarda. A written informed consent was obtained from all subjects.

**Figure 1.** CONSORT statement flow diagram.

#### *2.2. Clinical Procedures*

Patients underwent a fasting venous blood sampling and a full clinical examination, with determination of height, body weight, waist circumference, heart rate, and systolic and diastolic blood pressure (SBP, DBP). Bioelectric impedance analysis (ViScan device-Tanita Inc., Tokyo, Japan) was used to estimate % abdominal fat mass (BIA (%)) and % visceral fat rating (VFR (%)), according to reported procedures [12]. Plasma samples were immediately separated by centrifugation, and aliquots were immediately stored at −20 ◦C. In the present analysis, based upon the study reported in [18], we evaluated basal and postintervention circulating oxysterol levels. CV biomarkers (TC, non-HDL-C, triglycerides (TG), HDL-C, apolipoprotein (apo)AI, apoB, lipoprotein(a) (Lp(a)), proprotein convertase

subtilisin/kexin type 9 (PCSK9)) from this study were used for correlation analysis [18]. Data retrieval, analysis, and the preparation of the manuscript were solely the responsibility of the authors.

#### *2.3. Immunometric and Biochemical Assays*

In all blood samples, TC, HDL-C, TG, apoAI, apoB, Lp(a), fasting plasma glucose (FPG), uric acid were measured according to a standard automated clinical procedure (Cobas system, Roche, Italy). LDL-C was calculated according to the Friedewald formula. Non-HDL-C was calculated as TC minus HDL-C. Enzyme-linked immunosorbent assay (ELISA) kits were used according to manufacturer's specifications to quantify fibroblast growth factor (FGF) 19, FGF21 and PCSK9 [20] (R&D System, Minneapolis, MN, USA). Oxidized LDL (oxLDL), and insulin were measured by ELISA kits (Mercodia, Sweden). The homeostasis model assessment of insulin resistance (HOMA-IR) index was calculated according to this equation: HOMA-IR = [fasting glucose (mg/dL) × insulin (mUI/L)/405].

#### *2.4. Determination of Serum Levels of Oxysterols*

Oxysterols were analyzed as previous described [21] using a Thermofinnigan GC-Q instrument supplied with an ion trap source. Oxysterols separation was obtained with an HP5 (Agilent, Lexington, MN, USA) capillary column 0.25 mm i.d., 0.25 μm film thickness, 30 m length, operating at 1 mL/min helium flow rate. Column temperature was programmed from 200 to 300 ◦C at 20 ◦C/min. Ions were recorded at *m*/*z* 353 for 19-hydroxycholesterol and *m*/*z* 462 for deuterated 27-OHC (internal standards), *m*/*z* 456 for 27- and 25-OHC and *m*/*z* 413 for 24-OHC. Endogenous hydroxysterol concentrations were calculated with a standard curve, prepared as described [21], and the peak area ratio (sterol/IS) found in the sample. Noteworthy, in the literature the same chemical compound is called both 26-OHC and 27-OHC [22]. This second term over time has become common and to prevent misunderstandings in this study the term "27-OHC" will be used [23].

#### *2.5. Statistical Analysis*

Sample size calculation. According to [18], a group sample size of 16 per arm achieves 80% power to detect a difference of 20 mg/mL in absolute changes (12 weeks-0 week) in LDL-C levels (mg/mL), between the null hypothesis that in both arms the means of change in LDL-C are 10 mg/mL and the alternative hypothesis that the mean of change in LDL-C in the treatment arms is −10 mg/mL [12]. The estimated group standard deviations were 25 mg/mL per arm, with a significance level of 5% using a two-sided two-sample *t*-test.

Results are shown as median and interquartile ranges (Q1 and Q3) for all parameters. Correlations between circulating oxysterols as well as oxysterols normalized for TC and several covariates were analyzed using a Pearson correlation coefficient. Oxysterol levels are expressed both as absolute values as well as oxysterol-to-TC ratios, in order to correct for differences in plasma TC concentration and for the evidence that sterols are transported by plasma lipoproteins, in line with the available literature [24].

Differences in median values between treatment and placebo arms at baseline were assessed by Wilcoxon-rank sum test. Distributions of percent changes in oxysterols and lipid levels from baseline (0 week) to the end of follow-up (12 weeks treatment) were compared between the placebo and the treatment groups with Wilcoxon rank sum tests. At the end of the follow-up timeframe, the difference in the median percent change observed in the treatment group minus the median percent change in the placebo group at that time was used to summarize the treatment effect. Similar comparisons of percent changes in lipid levels between arms used the same approach. All tests are 2-sided; *p* values 0.05 are considered statistically significant. Statistical analysis was conducted by using both SAS Software version 9.3 (SAS, Cary, NC, USA) and R Software version 3.6.2.

#### **3. Results**

#### *3.1. Study Population*

All subjects were in primary CV prevention and showed a moderate hypercholesterolemia. The data analysis was conducted on the 30/33 subjects who also completed the interventional study (Figure 1). The clinical and biochemical data suggest that this cohort showed normal body weight, BMI, waist circumference, BIA and VFR (Table 1). Median TC was 270 (246, 288) mg/dL and LDL-C was 179 (169, 195) mg/dL. TG, HDL-C, insulin sensitivity and blood pressure were in the reference range [25,26].

**Table 1.** Main clinical and biochemical characteristics of the whole study population and sorted in the two arms.


Data are median (Q1, Q3). BMI: body mass index, BIA: bioelectrical impedance analysis/abdominal fat mass, VFR: visceral fat rating, SBP: systolic blood pressure, DBP: diastolic blood pressure, HR: heart rate, TC: total cholesterol, LDL-C: low-density lipoprotein. cholesterol, HDL-C: high-density lipoprotein cholesterol, TG: triglycerides, apoAI: apolipoprotein AI, apoB: apolipoprotein B, oxLDL: oxidized LDL, 24-OHC: 24-hydroxycholesterol, 25-OHC: 25-hydroxycholesterol, 27-OHC: 27-hydroxycholesterol, Lp(a): lipoprotein (a), PCSK9: proprotein convertase subtilisin/kexin type 9, FPG: fasting plasma glucose, HOMA-IR: Homeostatic Model Assessment of Insulin Resistance, FGF: fibroblast growth factor.

#### *3.2. Analysis of Oxysterols in the Study Population*

The 24-OHC values (89 (73, 109) ng/mL) of the study cohort are reported in Table 1. They did not differ according to sex (males: 89.4 (73.5, 110.5) ng/mL; females: 89 (73, 109) ng/mL) (Figure 2A) and did not correlate with age (Table 2). The 24-OHC/TC

ratio was 34 (27, 41) ng/mg when considering the entire cohort (Table 1), without sex difference (males 37 (27, 43) ng/mg; females 31 (27, 38)) (Figure 2B) and no correlation with age (Table 2). Intriguingly, 24-OHC was positively correlated with TG (*p* = 0.004) and apoB (*p* = 0.012) and, after normalizing the values for TC, only the correlation with TG was still present (*p* = 0.024) (Table 2). The values of 25-OHC were 84.2 (60.5, 96) ng/mL (Table 1) and did not differ according to sex (males 84.7 (72, 101) ng/mL; females 71.5 (54, 89) ng/mL) (Figure 2A), nor they correlated with age (Table 2). 25-OHC/TC levels (29 ng/mg (25, 38) (Table 1) were not different according to sex (males 32 (26, 41) ng/mg; females 27 (18, 32) ng/mg) (Figure 2B) and did not correlate with age (Table 2). Interestingly, both 25-OHC and 25-OHC/TC showed a significant positive correlation with TG (*p* = 0.007 and *p* = 0.028) (Table 2). Moreover, 24-OHC and 25-OHC levels were significantly correlated (*p* = 0.0002) (Table 2). The 27-OHC values (183.5 ng/mL (152, 211)), shown in Table 1, significantly (*p* = 0.02) diverged between males (209 (173, 230) ng/mL) and females (167.7 (126, 193) ng/mL) (Figure 2A) and did not correlate with age (Table 2). 27-OHC/TC values (72 ng/mg (51, 80)) were also different (*p* = 0.008) according to sex (males 77 (61, 90) ng/mg; females 56 (46, 72) ng/mg (Figure 2B) and did not correlate with age. In addition, 27-OHC showed a positive correlation trend with TG (*p* = 0.056) and non-HDL-C (*p* = 0.065), while it was significantly correlated with creatinine (*p* = 0.017). 27-OHC/TC was negatively correlated with HDL-C (*p* = 0.006) and apoAI (*p* = 0.05), whereas it was positively correlated with abdominal circumference (*p* = 0.023) and VFR (*p* = 0.021) (Table 2). Lp(a) was correlated with 24-OHC/TC (*p* = 0.021), 25-OHC (*p* = 0.045) and 25-OHC/TC (*p* = 0.013). Moreover, PCSK9 levels were negatively correlated with 27-OHC/TC (*p* = 0.013) (Table 2).

**Figure 2.** (**A**) 24-hydroxycholesterol (24-OHC), 25-hydroxycholesterol (25-OHC) and 27-hydroxycholesterol (27-OHC) plasma levels; (**B**) 24-OHC/total cholesterol (TC), 25-OHC/TC and 27-OHC/TC. TC values (mean ± SD) were 250.5 ± 75.6 mg/dL for males and 257.9 ± 80 mg/dL for females. M, males; F, females. \* *p*-value < 0.01.

#### *3.3. Effect of Nutraceutical Treatment on Oxysterols Plasma Levels*

The participants of the cross-sectional study were also randomized to either nutraceutical combination or placebo (Figure 1). No differences between arms were found for the different variables, except age and apoB (Table 1). After nutraceutical intervention, compared to placebo, TC was significantly reduced (*p* < 0.0001; −16.7%), together with LDL-C (*p* < 0.0001; −25.7%), as previously reported [18] (please refer to this article for additional data on CVD biomarkers). After the normalization for TC (24-OHC/TC, 25-OHC/TC and 27-OHC/TC), oxysterol levels did not differ between the 2 groups. When considering the absolute value of circulating oxysterols, in the nutraceutical treatment arm 27-OHC concentrations were significantly (*p* = 0.008) decreased (−10.4%), whereas 24-OHC and 25-OHC levels did not change (Table 3).


**Table 2.** Correlation of circulating oxysterols levels and total cholesterol-normalized circulating oxysterols levels with clinical and biochemical characteristics of the study population.

Pearson correlation coefficient and *P*-value are reported for each correlation, except for Sex (dichotomic variable). BMI: body mass index, BIA: bioelectrical impedance analysis/abdominal fat mass, VFR: visceral fat rating, SBP: systolic blood pressure, DBP: diastolic blood pressure, HR: heart rate, TC: total cholesterol, LDL-C: low-density lipoprotein. cholesterol, HDL-C: high-density lipoprotein cholesterol, TG: triglycerides, apoAI: apolipoprotein AI, apoB: apolipoprotein B, oxLDL: oxidized LDL, 24-OHC: 24-hydroxycholesterol, 25-OHC: 25-hydroxycholesterol, 27-OHC: 27-hydroxycholesterol, Lp(a): lipoprotein (a), PCSK9: proprotein convertase subtilisin/kexin type 9, FPG: fasting plasma glucose, HOMA-IR: Homeostatic Model Assessment of Insulin Resistance, FGF: fibroblast growth factor.

**Table 3.** Determination of serum levels of 24-hydroxycholesterol (24-OHC), 25-hydroxycholesterol (25-OHC) and 27 hydroxycholesterol (27-OHC) and their ratio to total cholesterol (TC).


Data are shown as median (1st quartile, 3rd quartile), *p*-values are adjusted for age and apolipoprotein B.

#### **4. Discussion**

The present study was aimed at analyzing the relationship between circulating oxysterols, namely 24-, 25- and 27-OHC, with biomarkers related to atherosclerosis in subjects with moderate hypercholesterolemia. We also evaluated the effect of a nutraceutical combination containing *Bifidobacterium longum* BB536 and RYR and aimed to reduce hypercholesterolemia, on the circulating levels of these oxysterols. In our cohort of moderate hypercholesterolemic subjects, 24-OHC values were found to be within the range (33.2– 227.0 ng/mL) previously reported for different populations [4]. On the contrary, the values of 25-OHC were almost 3 fold higher compared to those reported (range 2.0–31.0 ng/mL) previously [4]. Together with data indicating that hypercholesterolemic males have significantly higher 25-OHC levels compared to healthy males [10], our findings suggest that this elevation may be peculiar for this condition. 27-OHC values in our population were within the previously reported 27-OHC range (43.6–196.0 ng/mL) [4]. Different measurement techniques (high-performance liquid chromatography–mass spectrometry (HPLC-MS) without derivatization, charge-tagging with HPLC-MS analysis, dimethylglycine derivatization followed by HPLC-ESI-MS and GC–MShigh-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS) and gas chromatography-mass spectrometry (GC–MS) analysis of oxys-terol trimethylsilyl derivatives), each having peculiar features might account for the variation range in oxysterols levels [27]. We found a significant correlation between the circulating levels of 24-OHC and 25-OHC. In this regard, CYP46, the enzyme mainly responsible for 24-OHC synthesis, was also shown to be capable of synthesizing 25-OHC (ratio: 4:1 (24-OHC:25- OHC)), in cell-based systems [28].

Interestingly, we observed that 27-OHC and 27-OHC/TC plasma levels were significantly higher in males than in females. These findings agree with previously reported data for 27-OHC [29], extending the observation to 27-OHC/TC. Circulating 27-OHC levels are lower in females than in males in both rodents and humans, and this is most likely related to the upregulation of CYP7B1 expression by estradiol and estrogen receptor activation [13,23]. Interestingly, 27-OHC itself has also been regarded has a peculiar selective estrogen receptor modulator (SERM), and has been shown to interfere with the atheroprotective activity of estrogens [30]. These findings may be relevant in the overall context of sex-related ASCVD risk and related response to drugs [31]. Moreover, experimental work indicates that 27-OHC also has an adverse impact on bone mineralization [32] and breast cancer proliferation [33].

In healthy subjects, 24-OHC and 27-OHC were found to correlate with TC, LDL-C and non-HDL-C [24]. In our cohort of moderate hypercholesterolemic patients, we observed a positive trend between plasma 27-OHC and non-HDL-C, but no correlations of oxysterols with TC and LDL-C, although 24-OHC levels positively correlated with apoB concentrations.

In our moderate hypercholesterolemic patients, a significant negative correlation was found between 27-OHC/TC and HDL-C and apoAI, its main lipoprotein component, circulating levels. This observation is in agreement with a previously reported inverse relationship between 27-OHC and HDL-C in normocholesterolemic subjects [34,35]. One possible explanation may be that 27-OHC, acting as liver X receptor (LXR) α ligand, upregulates the expression of cholesteryl ester transfer protein (CETP), which in turn transfers cholesteryl ester from HDL to other lipoproteins, leading to HDL-C reduction [34] and possibly to LDL-C increase [36]. It may be then speculated that the ineffective reverse cholesterol transport in individuals with very low levels of HDL-C may be compensated by this mechanism, which therefore could represent an alternative pathway in the context of the complex regulation of reverse cholesterol transport [37]. In our normo-triglyceridemic population, 24-, 25- and 27-OHC correlated with TG. While the 27-OHC and TG relationship has already been reported in healthy subjects [24], that between 24-, 25-OHC and TG is novel, to the best of our knowledge, and is still present upon normalization by TC

(24-, 25-OHC/TC and TG). This correlation could be the consequence of the known LXR modulation by these oxysterols [38] and the resulting stimulation of liver TG synthesis [39].

In the context of the studied population, featuring normal BMI and abdominal circumference, we observed that the 27-OHC/TC ratio positively correlated with abdominal circumference and VFR%. This finding is novel in the clinical setting, although the interrelationship between 27-OHC and adipose tissue has previously been addressed experimentally, leading to apparently controversial observations. In addition to circulate in serum, 27-OHC may also be locally produced by rodent and human adipocytes, where it may counteract adipogenesis [40]. 27-OHC content of white adipose tissue was negatively correlated with adipose mass in mice and exposure to 27-OHC suppressed intracellular TG accumulation by down-regulating lipogenic and adipogenic gene expression during adipocyte maturation of mouse 3T3-L1 cells [41]. However, in mice, 27-OHC administration has been shown to promote adipose tissue hyperplasia, independently from diet type, increasing visceral fat and local inflammation [42]. In light of the relevance of dysfunctional visceral and ectopic adipose for ASCVD [43,44], this intriguing connection requires further clarification.

To our knowledge, no data are available on the effects of hypocholesterolemic nutraceutical treatments on circulating oxysterol levels. Since, in this field, several nutraceutical combinations include RYR, whose main active component is monacolin K, notoriously structurally identical to the statin lovastatin, we may consider a comparison with the available data regarding the impact of statins treatment on 24-, 25- and 27-OHC in subjects with moderate hypercholesterolemia [10,45]. After nutraceutical intervention, 24-OHC level and 24-OHC/TC ratio were unchanged, differently with the reducing effect of simvastatin (80 mg/day) or atorvastatin (40 mg/day) [11,45]. The 25-OHC concentration and 25-OHC/TC ratio were also not affected in both placebo and active groups. Previously reported effects of statins showed decreased 25-OHC concentrations in hypercholesterolemic patients [10,46]. In agreement with the effect of simvastatin (80 mg/day) and atorvastatin (40 mg/day) [11], in our study, 27-OHC levels were significantly reduced in the nutraceutical group, whereas the 27-OHC/TC ratio was not different between arms. 27-OHC levels were also found to be decreased after treatment with atorvastatin or rosuvastatin in subjects with familial hypercholesterolemia or familial combined hyperlipidemia [47]. Due to the similarity of action of RYR extracts and statins, the observed 27-OHC reduction in our study may be the result of cholesterol synthesis inhibition, as also supported by the unchanged 27-OHC/TC ratio. Interestingly, such 27-OHC reduction may also contribute to the observed LDL-C decrease via downregulation of the CETP-pathway. As the nutraceutical combination used here, in addition to RYR, also contains the probiotic *Bifidobacterium longum* BB536, niacin, and coenzyme Q10, one should not exclude some contribution of these components to the effects on the observed reduction in 27-OHC level. One may hypothesize that *Bifidobacterium longum* BB536 may possibly contribute to this reduction by means of its biliary salt hydrolase activity, taking place in the ileum and consequently interfering with the enterohepatic circulation of cholesterol [18].

The evaluation of oxysterol levels after treatment with this nutraceutical combination provides further information on the safety of this product. Oxysterols seem to have a pathogenic role in hyperlipidemia and atherosclerosis both via modulating numerous systemic functions as well as with local actions, and the absolute reduction of 27-OHC concentration may result beneficial, in the context of this cohort of hypercholesterolemic subjects. Elevated circulating 27-OHC may have detrimental effects on the cardiovascular system through multiple mechanisms (SERM activity, LXRα and β ligand), promoting vascular inflammation, which is critically involved in atherogenesis [42,48–50]. The additional contribution of 27-OHC and CYP27A1, responsible for its synthesis, which are abundantly present in atherosclerotic plaques, needs further studies [29,51,52].

The findings of the present study may be relevant in terms of long-term safety in consideration of the potentially detrimental role of increased 24-, 25- and 27-OHC also in the context of neurodegenerative diseases such as mild cognitive impairment and dementia [53], as well as bone mineralization and breast cancer [54–56].

The main strengths of this study include (1). the extensive exploration of the relationships between 24-, 25- and 27-OHC with a relevant set of CVD risk biomarkers in subjects with moderate hypercholesterolemia, highlighting a series of novel correlations, and (2). the first evaluation of the impact of a nutraceutical combination, designed for the control of hypercholesterolemia, on the circulating levels of these oxysterols. The present study has some limitations. The lack of a control group, useful for comparison, is an intrinsic limitation of the cross-sectional study. Therefore, several observations cannot be extended to healthy subjects. A limitation of the interventional study is also that the dietary intake of the volunteers randomized to either placebo or nutraceutical intervention was not recorded.

Due to their relevant biological effects, the measurement of 24-, 25- and especially 27-OHC may be useful even when assessing the long-term safety of hypocholesterolemic treatments (statins, ezetimibe, bempedoic acid and PCSK9 inhibitors), indicating the need for further larger studies.

#### **5. Conclusion**

In conclusion, this study adds novel information on this hypocholesterolemic nutraceutical combination, regarding its efficacy and safety, according to oxysterol profile.

**Author Contributions:** Conceptualization, P.M. and S.C; methodology, M.D.P.; software, C.P.; validation, M.B. and G.M.; formal analysis, M.D.P.; investigation, M.B.; resources, M.B.; data curation, E.O.; writing—original draft preparation, P.M. and S.C.; writing, review and editing, A.L.C.; visualization, L.C.; supervision, A.L.C.; project administration, P.M.; funding acquisition, P.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was supported by unrestricted grants to Centro Dislipidemie (ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy) and Università degli Studi di Milano (Milan, Italy) from Montefarmaco OTC S.p.A. (Bollate, MI, Italy). The founding sponsors had no role in the design of the study, in collection, analyses, or interpretation of data, in the manuscript writing, and in the decision to publish the results.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee "Milano Area C" at ASST Grande Ospedale Metropolitano Niguarda (protocol code 598-112015 – 27 November 2015).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy reasons.

**Acknowledgments:** The critical revision of the manuscript by G.D. Norata is gratefully acknowledged.

**Conflicts of Interest:** S.C., C.P., E.O., M.D.P., M.B., G.M. and L.C. have no conflict of interests. A.L.C. has received honoraria, lecture fees, or research grants from: Akcea, Amgen, Astrazeneca, Eli Lilly, Genzyme, Kowa, Mediolanum, Menarini, Merck, Pfizer, Recordati, Sanofi, Sigma Tau, Medco, Amryt. P.M.: has received honoraria, lecture fees, or research grants from: Montefarmaco OTC.

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### *Review* **Phytosterols, Cholesterol Control, and Cardiovascular Disease**

#### **Andrea Poli 1,\*, Franca Marangoni 1, Alberto Corsini 2,3, Enzo Manzato 4, Walter Marrocco 5, Daniela Martini 6, Gerardo Medea <sup>7</sup> and Francesco Visioli 8,9**


**Abstract:** The use of phytosterols (or plant sterols) for the control of plasma cholesterol concentrations has recently gained traction because their efficacy is acknowledged by scientific authorities and leading guidelines. Phytosterols, marketed as supplements or functional foods, are formally classified as food in the European Union, are freely available for purchase, and are frequently used without any health professional advice; therefore, they are often self-prescribed, either inappropriately or in situations in which no significant advantage can be obtained. For this reason, a panel of experts with diverse medical and scientific backgrounds was convened by NFI—Nutrition Foundation of Italy—to critically evaluate and summarize the literature available on the topic, with the goal of providing medical doctors and all health professionals useful information to actively govern the use of phytosterols in the context of plasma cholesterol control. Some practical indications to help professionals identify subjects who will most likely benefit from the use of these products, optimizing the therapeutic outcomes, are also provided. The panel concluded that the use of phytosterols as supplements or functional foods to control Low Density Lipoprotein (LDL) cholesterol levels should be preceded by the assessment of some relevant individual characteristics: cardiovascular risk, lipid profile, correct understanding of how to use these products, and willingness to pay for the treatment.

**Keywords:** phytosterols; plant sterols; cholesterol; cardiovascular disease; supplements; functional foods

#### **1. Introduction**

The use of supplements or functional foods to keep plasma cholesterol concentrations under control is growing steadily in European countries [1–3] Among these products, phytosterols (or plant sterols) have recently gained traction because their cholesterollowering efficacy, within the frame of a healthy lifestyle, is acknowledged by authoritative guidelines [4] and, among others, by the European Food Safety Authority (EFSA) [5,6].

In the European Union, such products, formally classified as "food", can be freely purchased by the public under self-prescription; it is reasonable to believe that, if used after a professional prescription and under medical control, the appropriateness of their use and, consequently, their efficacy in improving plasma cholesterol concentrations and cardiovascular risk would significantly improve.

For this reason, NFI—Nutrition Foundation of Italy—has convened a group of experts with diverse medical and scientific backgrounds to critically evaluate and summarize the

**Citation:** Poli, A.; Marangoni, F.; Corsini, A.; Manzato, E.; Marrocco, W.; Martini, D.; Medea, G.; Visioli, F. Phytosterols, Cholesterol Control, and Cardiovascular Disease. *Nutrients* **2021**, *13*, 2810. https:// doi.org/10.3390/nu13082810

Academic Editor: Lindsay Brown

Received: 19 July 2021 Accepted: 13 August 2021 Published: 16 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

literature available on the topic, and to provide some practical indications to help health professionals identify persons who will most likely benefit from the use of phytosterols. The main goal of this effort is to entrust doctors who perform clinical activities and all health professionals with a proper use of these products, to improve cardiovascular prevention in the population.

#### **2. Phytosterols' Chemistry**

Phytosterols are fat-soluble compounds belonging to the triterpene's family, present in most plant cells where they contribute to membranes structure and stability. They are characterized by a tetracyclic structure, with a side chain in position 17 of the D ring [7]. Their structure is very similar to that of cholesterol, which is by far the most abundant sterol in animal cells, where it plays a similar structural role. Phytosterols differ from cholesterol in the side chain bound in their C-17 position; sitosterol, as an example, has an ethyl group linked in C-24 of the side chain, while campesterol has a methyl group in the same position, which is empty in cholesterol. Phytostanols are 5alpha-saturated derivatives of phytosterols [8]. Several hundred different phytosterol molecules have been identified in plant cells; the most common ones are beta-sitosterol, campesterol, stigmasterol, brassicasterol, and avenasterol [9,10].

The food content in phytosterols is highest in oily fruit, oil seeds, and in the oils obtained from them [11,12]. In particular, rapeseed oil, wheat germ oil, and corn oil are the oils richest in phytosterols, whereas among the various types of oily fruit the highest content is found in pistachios [13]. Phytosterols are also present in legumes and cereals, whilst fruit and vegetables contain much lower quantities. In general, the concentration of total phytosterols in vegetables varies from a few milligrams or tens of milligrams per 100 g of fruit and vegetables up to over 1000 mg per 100 g in some vegetable oils, with large differences among different foods [14].

In European countries, the overall dietary intake of phytosterols is around 250–400 mg/day, with a high variability [15]: a value quite similar to dietary cholesterol intake. The dietary intake may vary according to the prevalent dietary pattern; the highest content has been found in vegan diets (up to 500 mg/day). The most abundant dietary phytosterol is sitosterol (about 60–70% of total phytosterols in the diet), followed by campesterol (16%) and stigmasterol (10%) while sitostanol, campestanol, and Δ5-avenasterol collectively contribute <10% [16].

#### **3. Human Metabolism and Metabolic Effects of Phytosterols**

Due to their lipophilicity, phytosterols ingested with foods or supplements or enriched/functional foods, are absorbed by the human intestine after incorporation into the so-called "mixed micelles". These micelles derive from the emulsification of dietary fats by bile salts, and allow the entry of phytosterols into the enterocytes through a well characterized membrane transport protein called Niemann-Pick C1—Like 1 (NPC1L1). Most of absorbed phytosterols are immediately re-excreted in the intestinal lumen by efflux transporters of the ATP-binding cassettes (ABC) family, known as ABCG5 and ABCG8 [17]. These metabolic pathways are summarized in Figure 1 [18,19]. Limited amounts of phytosterols, instead esterified within enterocytes, incorporated in chylomicrons, and eventually captured by the liver, are to a large extent secreted into the bile through the ABCG5/G8 transporters present in the biliary pole of hepatocytes.

Plasma concentrations of phytosterols are, consequently, lower (usually by two orders of magnitude) than those of cholesterol, essentially due to the limited intestinal absorption (less than 5% of plant sterols and less than 0.5% of stanols are absorbed and enter the systemic circulation [11], versus about 50–60% of dietary cholesterol [20]) and to the rapid hepatic clearance through the bile.

**Figure 1.** Main metabolic pathways of cholesterol and phytosterol in enterocytes. P, phytosterols.; C, cholesterol; CE, cholesteryl esters; ACAT, acylCoA cholesterol acyltransferase; NPC1L1, Niemann-Pick C1-Like 1; ABCG5, ATP-binding cassette G5; ABCG8, ATP-binding cassette G8; TG, triglycerides; B48, Apolipoprotein B-48. Modified from [18] and [19].

As cholesterol (deriving from ingested food or from the bile) is absorbed from the gut through the same pathway used by phytosterols, phytosterols compete with it for incorporation in the mixed-micelles and subsequent absorption in enterocytes, through the NPC1L1 transporter. Hence, cholesterol fractional absorption declines along with increasing amount of phytosterols present in the gut. The inhibition of cholesterol absorption by phytosterols ranges from about 5% for daily intakes of 300–400 mg (typical of most diets) up to 35–40% for intakes between 1500 and 2000 mg per day, which can only be achieved using enriched functional foods or specific supplements. In addition, phytosterols can also limit the absorption of cholesterol by directly co-crystallizing with cholesterol itself in the intestinal lumen and facilitating its elimination via the fecal route.

#### **4. Effects of Phytosterols on Low Density Lipoprotein (LDL) Cholesterol: Characteristics and Clinical Relevance**

The reduction in the amount of cholesterol absorbed from the intestine and reaching the liver through the chylomicron pathway triggers both a greater endogenous synthesis of cholesterol and a greater uptake of plasma LDL by hepatocytes, to maintain cholesterol homeostasis. The greater clearance of circulating LDL cholesterol yields the desired reduction in its plasma concentration. Such reduction is around 2–3% for the aforementioned dietary intakes of phytosterols (300–400 mg/day) [21] and reaches an average of 9% for supplementary dosages between 1500 and 2000 mg per day [22]. The effect can be as strong as a 12–12.5% reduction for dosages up to 3 g/day and tends to plateau for higher intakes [5].

Recent studies have confirmed such effects of phytosterols on plasma total and LDL cholesterol levels. Meta-analyses of published trials [23,24] indicate that the effect of the intake of phytosterols on plasma LDL cholesterol levels in humans falls within the range indicated by EFSA (Commission Regulation (EU) No 384/2010).

The efficacy of plant sterols on LDL cholesterol is independent of the initial LDL cholesterol concentrations; therefore, it can be useful in subjects with both low and high baseline LDL cholesterol levels [25]. Even in the presence of heterozygous familial hypercholesterolemia, the use of phytosterols can help reduce plasma LDL levels, as observed in, e.g., children [26].

High Density Lipoprotein (HDL) cholesterol is generally not significantly affected by phytosterols; triglycerides plasma levels are reduced to a minor extent, but the effect is larger when their levels exceed 150 mg/dL [27].

Because, as described above, the lipid-lowering effects of phytosterols are due to a competitive inhibition, such effects rapidly taper off upon discontinuation of intake and disappear after 7–10 days from the last dose of supplements/functional foods [6].

According to some authors [8], stanols are slightly more effective than the corresponding sterols, but a meta-analysis on the subject did not identify significant differences in the effect on LDL cholesterol levels between the two groups of molecules [23]. Furthermore, the effects do not appear to be influenced by the chemical form (free or esterified) in which sterols and stanols are ingested [28].

Experimental studies suggested that, perhaps due to the reduction of LDL cholesterol, phytosterols may perform a modest anti-inflammatory action. Nevertheless, according to a meta-analysis, regular intake of food enriched with phytosterols did not significantly impact levels of biomarkers of low-grade inflammation in obese subjects [29]. An antiinflammatory effect, on the other hand, might also derive, at least in part, from an interaction between phytosterols and microbiota, improving the state of dysbiosis associated low-grade inflammation [30]. There is also some in vitro and in vivo experimental evidence suggestive of modulatory roles of phytosterols, namely through a reduction of selected bacterial species [10,31].

Endothelial function, evaluated as flow mediated dilation, would also improve after a treatment with phytosterols, but this is also controversial. Mechanistically, this effect could explain the mild reduction in blood pressure found in a recent meta-analysis [32].

Unfortunately, probably because of the large number of subjects needed and the challenge of controlling diets for a very long period, no data deriving from formal randomized clinical trials are available allowing to translate this well described effect of phytosterols on plasma LDL cholesterol levels into measurable direct clinical effects on cardiovascular morbidity and mortality [33].

Although such absence of clinical trials showing that phytosterols intake can reduce the incidence of clinical endpoints, such as myocardial infarction or coronary deaths, needs to be acknowledged, it is also necessary to remember that the accrued evidence clearly shows that lowering cholesterol concentrations by any means, e.g., via diet, ileal by-pass, or drugs with different mechanisms of action, is always accompanied by a proportional reduction in cardiovascular risk. Hence, both European Atherosclerosis Society (EAS) and EFSA [4,5] state that the plasma LDL cholesterol-reducing effects of phytosterols will proportionally reduce cardiovascular risk and related coronary events.

Interestingly, the selective effect of phytosterols on cholesterol absorption may have some positive preventive consequences.

It is well known that the balance between the rate of intestinal absorption or of hepatic synthesis of cholesterol in driving plasma LDL cholesterol levels may be different according to individual characteristics: in some individuals (often called "absorbers") a prevailing absorbing pattern from the gut can be observed, while in other subjects (often called "synthesizers"), hepatic synthesis is largely prevailing.

Subjects with genetic variants of NPC1L1 that limit cholesterol absorption have a much lower cardiovascular risk than their genetic counterparts with normal NPC1L1 activity, even if their cholesterol concentrations are only slightly lower [34]. Observational studies [35], and a meta-analysis [36], indeed, indicate that cardiovascular risk is higher in absorbers than in synthesizers, even if their plasma LDL cholesterol levels are comparable. Patients with chronic renal failure are usually absorbers and their higher cardiovascular risk and mortality seem to be related at least in part to their absorptive pattern [37,38].

This higher cardiovascular morbidity observed in absorbers as compared with synthesizers might be explained by the observation that the NPC1L1-mediated intestinal

cholesterol absorption is poorly selective and takes up also molecules structurally similar to cholesterol, but potentially more atherogenic, such as oxysterols. Oxysterols are strongly atherogenic in experimental models and the blockage of NPC1L1 prevents the vascular damage exerted by these molecules [39].

These data, altogether, suggest that if an equal degree of LDL lowering is achieved, the effect obtained through inhibition of cholesterol absorption might be more advantageous than that obtained inhibiting cholesterol synthesis; the clinical benefits of phytosterols on the cardiovascular risk, consequently, might be larger than that solely expected by their impact on LDL cholesterol levels.

Preliminary data also suggest a possible preventive role of phytosterols in relation to the risk of some cancers and obesity, as well as a possible immunomodulatory role [4,40]. These associations, on the other hand, are more difficult to interpret from a mechanistic viewpoint, and could consequently be non-causal; they require further investigation.

#### **5. Variables Affecting the Cholesterol-Lowering Effect of Phytosterols**

A significant variability can be observed in the plasma cholesterol response of different individuals to phytosterols treatment.

The cholesterol-lowering effect of phytosterols supplementation in subjects with a typically synthetic pattern (for example, obese subjects, especially if insulin-resistant or frankly diabetic) will be smaller than that of persons with a profile more shifted towards the absorbing type (normal weight subjects with normal insulin sensitivity) [41].

The ApoE isoforms profile also appears to influence the efficacy of phytosterols, which may be higher in subjects with the E4 variant (at increased cardiovascular and cognitive decline risk) and lower in subjects with the more common E3/E3 isoform [42].

The possible effects of polymorphisms of other genes potentially influencing the plasma lipid response to the use of these products are also currently being evaluated.

On the other hand, age and sex do not appear to significantly affect the cholesterollowering response to phytosterols (which is perhaps slightly larger in males) [43].

A large number of studies has also considered the possible effect of variables that could affect the effectiveness of phytosterols supplementation on plasma LDL cholesterol concentrations [25,44,45]. In particular, the effect of the type of matrix (dairy products vs. other items, high fat vs. low fat foods, solid vs. liquid products), or of the type of administration (supplements vs. foods containing phytosterols), or of the method of supplementation (single dose vs. multiple doses), and the specific molecules used (sterols vs. stanols) have been considered [43].

In general, the data show greater efficacy when phytosterols are presented in solid rather than liquids foods [46]. However, this difference mainly develops at high dosages, whereas at the commonly used ones it appears to be negligible. A possible explanation of the lower efficacy of liquid foods can be linked to the faster gastric emptying, which results in a swifter transit time in the gastrointestinal tract where phytosterols play their cholesterol-lowering role [23].

In spite of the differences between solid and liquid matrices, the consumption of phytosterols incorporated in different foods does not appear to significantly influence their effects on plasma lipids. For example, the intake of phytosterols in milk-based or cereal-flour-based matrices has comparable effects on LDL cholesterol levels. Similarly, the cholesterol-lowering effects did not differ when comparing products rich in fat and non-fat foods [25,28].

Functional foods enriched with phytosterols and supplements based on phytosterols in capsules or tablets appear to have a similar effect on plasma LDL cholesterol concentrations [45]. Likewise, daily consumption in a single dose seems to be equally effective as the same quantity divided into three doses with meals. On the other hand, taking phytosterols at the end of one of the main meals, as compared with during fasting, amplifies the effect on plasma LDL cholesterol levels; consumption in a single dose at breakfast (especially after a small breakfast) is associated with a lower (less 30%) effect [47]. The explanation for this

difference probably lies in the greater presence, after a meal, of cholesterol of food or biliary origin in the intestine, with which phytosterols can compete limiting its absorption [48]. Interestingly, since the cholesterol present in the intestine is largely, i.e., at least 75% of biliary and not food origin, supplements or foods enriched in phytosterols are also effective in vegetarians and vegans, who introduce low or negligible amounts of cholesterol with the diet.

Finally, no clear differences between supplementations with sterols or stanols, or comparing phytosterols in free or in esterified form have been described. According to a recent study, however, the extent of the achievable reduction depends on the specific mixture of sterols used and would increase (by a few percentage points) if at least 80% of the phytosterols used are composed of beta-sitosterol or the corresponding stanol [24].

#### **6. Regulatory Framework**

Some components of the regulatory framework of foods and food ingredients (as well as supplements and functional foods) aimed at controlling LDL cholesterol levels are relevant for their proper use and for a correct understanding of the interactions between manufacturers, medical doctors, and consumers.

In the European Union, phytosterols are classified among foods and are subject to the comprehensive food legislation. Communication of beneficial effects in relation to health and nutrition, on the product label or in the advertising of foods (as well as for food supplements), is defined by Regulation (EC) No 1924/2006 and is limited to "function health claims", pursuant to article 13(5) (for example: "product x contributes to the maintenance of normal blood cholesterol levels") and to "risk reduction claims" pursuant to art. 14(1)(a) of the same Regulation, on reducing a risk factor in the development of a disease (for example: "product y has been shown to lower/reduce blood cholesterol. High cholesterol is a risk factor in the development of coronary heart disease").

It is interesting to underline that only phytosterols (at a dose of 1.5–3 g/day) and beta-glucans (at a dose of 3 g/day) can benefit from a European Commission-authorized claim pursuant to art. 14(1)(a), which allows us, in the communication to the public, to refer to a cholesterol-lowering effect. Furthermore, for phytosterols, the precise magnitude of the effect to be expected has been defined (specifically from 7 to 10% if foods ensure a daily intake of 1.5–2.4 g/day and from 10 to 12.5% for 2.5–3 g/day), and the duration to obtain the effect, "in 2 to 3 weeks", must be specified.

This information can be used in promoting or advertising phytosterol enriched foods and supplements; notably, as already mentioned, it is reasonable to assume that a professional support, where possible medical, to the use of these formulations, formally classified as "food" could significantly improve the appropriateness of their use [49,50].

#### **7. How to Identify Optimal Candidates for the Use of Phytosterols to Reduce LDL Cholesterol Levels**

The recent Guidelines of the European Societies of Cardiology and Atherosclerosis (ESC/EAS) on treating cholesterol levels to lower cardiovascular risk are based on principles that can be considered the foundations of a correct approach to hypercholesterolemic patients [51]. These principles can be summarized as follows: (1) The correlation between increasing plasma LDL cholesterol and increasing cardiovascular risk is continuous; (2) there are no plasma levels below which a reduction in LDL cholesterol becomes ineffective, not being accompanied by a reduction in the risk of cardiovascular clinical events; (3) the decision to treat a patient, on the other hand, needs to be based on an estimate, as accurate and complete as possible, of his/her global cardiovascular risk, i.e., the probability to incur in a fatal or not fatal cardiovascular events over the following years; (4) in primary prevention, the future risk of cardiovascular events can be estimated using the SCORE algorithm [52]. This estimate can then be integrated by information regarding the specific characteristics of the patient, such as personal and family history, presence of other "classic" and "non-classic" risk indicators—socioeconomic status, level of individual stress, exposure to air pollution, sleep quality, etc.; and (5) as the estimated risk increases, therapeutic intervention on plasma cholesterol levels must progressively become more and more aggressive to reach lower LDL cholesterol values (target).

The aforementioned ESC/EAS Guidelines [51] recognize phytosterols with a significant and dose-dependent capacity to reduce LDL cholesterol (level of evidence A) without relevant effects on plasma HDL cholesterol and triglycerides levels. The Guidelines, also in light of the absence of significant side effects associated with their use [53], suggest considering phytosterols at doses up to 2 g/day after the main meal, leading to an average reduction of LDL cholesterol ranging from 7 to 10% in: (a) people with high cholesterol, low or intermediate overall cardiovascular risk, with no indication for drug treatment; or (b) patients at high or very high risk who do not reach their therapeutic goal in terms of LDL cholesterol despite treatment with statins (or who do not tolerate statins) to whom phytosterols can be administered in addition to drug therapy; or (c) adults and children (over 6 years of age) with familial hypercholesterolemia, within the Guidelines' framework.

As anticipated, although no data are available on the direct clinical effects of phytosterols on cardiovascular morbidity and mortality, clear evidence shows that lowering cholesterol concentrations by any mechanisms is always accompanied by a proportional reduction in cardiovascular risk [51].

Furthermore, as described in the EAS Guidelines [51], long-term monitoring studies indicate that phytosterols have a favorable safety profile which justifies their use as cholesterol-lowering agents both alone and in combination with drug therapy.

In this context, it is opinion of the expert group signing this document that the use of phytosterols as supplements or functional foods can be considered mainly in two different cases:

(1) People under the age of 40 years: in these subjects, estimating cardiovascular risk using the SCORE algorithm is formally not possible. Once patients with genetic hypercholesterolemia or with a previous cardiovascular event, whose plasma cholesterol levels must be treated according to the appropriate Guidelines indications, have been excluded, the cardiovascular risk of these people can be considered low by definition. However, on the basis of a thorough clinical evaluation including an accurate estimate of individual risk characteristics, a physician may decide to intervene on the cardiovascular risk of individual subjects by lowering their cholesterol; in these population groups, the use of a drug should be considered as off-label and, consequently, the use of supplements or functional foods is a valid alternative. Because, in these cases, the therapeutic goal for LDL cholesterol is set at 115 mg/dL, the optimal clinical target of phytosterols, as monotherapy, is represented by people who, following a correct diet, have a basal LDL cholesterol equal to or less than 130 mg/dL (Figure 2, flow chart A).

In individuals with higher plasma basal LDL cholesterol levels, the clinician may consider suggesting a combination of phytosterols with other food supplements indicated for cholesterol control.

(2) People over 40 years of age: in this age range, the prescription of the use of functional foods or supplements based on phytosterols should be considered for people with low or moderate risk, i.e., below 1% or in the 1–5% range at 10 years, respectively. For persons with a risk below 1% at 10 years, the target value for LDL cholesterol is set at 115 mg/dL. They can be also be treated as indicated in Figure 2, flow chart A.

For persons at moderate risk (1–5% at 10 years), the target value for LDL cholesterol is set at 100 mg/dL; phytosterols can be used in these persons as monotherapy when basal cholesterol levels are <110 mg/dL (Figure 3, flow chart B). Again, in individuals with higher plasma basal LDL cholesterol levels the clinician may consider to suggest a combination of phytosterols with other food supplements indicated for the cholesterol control.

**Figure 2.** Flowchart A. Patient <40 years old or >40 years old, but with global cardiovascular risk <1%. This flow-chart is not appropriate for patients with genetic disorders of lipoprotein metabolism or with manifest cardiovascular disease, who need to be treated according to the Guidelines.

**Figure 3.** Flowchart B. Patient ≥40 years old with global cardiovascular risk 1–5%. This flow-chart is not appropriate for patients with genetic disorders of lipoprotein metabolism or with manifest cardiovascular disease, who need to be treated according to the Guidelines.

The expected LDL reductions discussed in previous paragraphs, on the other hand, reflect the average efficacy of phytosterols. Individual responses may also be significantly different and the reduction of LDL cholesterol concentrations that can be achieved can be larger, e.g., among absorbers.

The use of supplements or functional foods enriched in phytosterols in subjects with higher cardiovascular risk, in association with other drugs with a complementary mechanism of action, e.g., statins, as outlined by the ESC/EAS Guidelines mentioned above, can be considered after a careful personalized evaluation.

#### **8. Side Effects of Phytosterols Use**

The use of supplements or foods enriched in phytosterols, within the limit of 1.5–3.0 g per day, is not associated with relevant side effects [4,18].

Intestinal absorption of some carotenoids is moderately reduced by phytosterol intake, bringing their plasma levels to the low end of the oscillation range physiologically observed

throughout the year (maximum in spring and summer and minimum in late winter). This reduction can be easily compensated by adopting a diet rich in these compounds, i.e., rich in colorful fruits and vegetables.

Some authors have proposed that an increase in plasma levels of phytosterols may represent a risk factor for cardiovascular events [54]; however, it is likely that, actually, their increase in circulating concentrations is rather an indicator of a high efficiency of the cholesterol absorption pathway, potentially atherogenic as previously discussed, and not a direct causal factor of atherosclerotic risk. In fact, no accumulation of phytosterols is observed in the tissues of subjects who take the recommended dosages of these compounds.

Post-marketing surveillance studies did not report any significant untoward effect of phytosterol use [55,56]. Of note, in patients with homozygous sitosterolaemia (in which the ABCG5 and/or ABCG8 transporters are not functional), the dietary intake of phytosterols greatly increases cardiovascular risk: the prevalence of this condition is, however, extremely low (about 1:10,000,000 of subjects) [57].

#### **9. Use of Phytosterols in Addition to Other Supplements and Drugs**

Other supplements and functional foods (or nutraceuticals), with different characteristics and mechanisms of action, are used worldwide for plasma LDL cholesterol control. Knowing the mechanisms underlying the effect of the aforementioned active ingredients on LDL cholesterol allows for their rational combinations, with the ultimate aim of optimizing their efficacy and safety.

For example, monacolin K, contained in fermented red rice, is chemically identical to lovastatin and inhibits the hepatic synthesis of cholesterol (the European Commission authorizes the claim "contributes to the maintenance of normal blood cholesterol levels" for products that provide at least 10 mg of monacolin K per day) [58]. The action of berberine is more articulated, and may include partial inhibition of PCSK9.

The mechanism of action of beta-glucans is quite similar to that of phytosterols. In this case, the European Commission has authorized the claim of reduction/maintenance of blood cholesterol levels for a daily intake of 3 g of beta glucans from oats, oat bran, barley, barley bran, or from mixtures of these sources [59].

A combination of phytosterols (inhibitors of cholesterol absorption) and statins (inhibitors of cholesterol synthesis) can be useful in subjects with more markedly altered lipid patterns (Figure 4) [60]. Phytosterols can, in fact, neutralize the compensatory increase in intestinal cholesterol absorption induced by statin. This combination therapy has been proposed based on the observation that the effect of phytosterols is additive to that of a diet low in saturated fat and statins [22,61]. Its efficacy is shown by a meta-analysis of 15 randomized clinical trials, including more than 500 subjects, which provides evidence that phytosterol enriched diets additionally lower total cholesterol and LDL cholesterol levels beyond that achieved by statins alone [60].

Theoretically, such effect might contribute to reduce the residual risk observed in patients treated with statins, which might be in part explained by the increased absorption pattern observed in these patients, but such interpretation must be considered merely speculative.

An additive effect on plasma LDL cholesterol levels can also by hypothesized for phytosterols and berberine [62]; the association of phytosterols with fiber and beta-glucans should be considered, on the opposite, less rationale [63]. Probably for the same reason, the evidence of add-on effects of phytosterols and ezetimibe is limited [64].

**Figure 4.** Combination of phytosterols with other food supplements or drugs active on plasma cholesterol levels. Blue (continuous) arrows: appropriate combination. Red (dotted) arrows: less appropriate combinations.

#### **10. Conclusions and Practical Suggestions**

Functional foods or supplements containing phytosterols are effective in controlling plasma LDL cholesterol levels if used appropriately.

These products must be taken on a daily basis; their cholesterol-lowering effect is rapid (noticeable after about three weeks); however, it is maintained only if the intake of phytosterols is routine. As mentioned above, the correct use of phytosterols induces an average reduction in plasma LDL cholesterol of 9–10%, which can reach up to 12.5% with higher dosages [65]; this reduction is added to that obtainable through appropriate diet and may be greater in some people, i.e., the absorbers.

Functional foods or supplements containing phytosterols should be taken in a single daily dose at the end of one of the main meals (lunch or dinner). Their administration on an empty stomach or after a small breakfast is not recommended [47].

The use of phytosterols is usually well tolerated, with no significant side effects. During the treatment, it is advisable to increase the intake of colored fruit and vegetables, especially the yellow, orange, and red ones.

The decision to propose the use of phytosterols as supplements or functional foods to control LDL cholesterol levels should be made by a physician or a qualified health professional after an assessment of individual cardiovascular risk, lipid profile, correct understanding of how taking these products, and willingness to pay for the treatment.

**Author Contributions:** Conceptualization, A.P. and F.M.; writing—original draft preparation, review and editing, A.P., F.M., A.C., W.M., D.M., E.M., G.M., F.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** The preparation of this paper has been made possible by an unrestricted grant from Danone S.p.A. The sponsor had no role in the preparation and finalization of the manuscript, and in the decision to publish it.

**Conflicts of Interest:** A.P. and F.M. are the Chairman and Head of Research, respectively, of NFI— Nutrition Foundation of Italy, a non-profit organization partially supported by Italian and non-Italian Food Companies. All other authors declare no conflict of interest associated with this publication.

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