The present study is a lipoprotein analysis from a previous double-blind, randomized, and placebo-controlled trial that utilized policosanol (Raydel^® Cuban Policosanol, 20 mg/day) or a placebo that was taken for 12 weeks by healthy Japanese participants (15). Cuban policosanol was defined as a genuine policosanol with a specific ratio of each ingredient (16): 1-tetracosanol (C24H49OH, 0.1–20 mg/g); 1-hexacosanol (C26H53OH, 30.0–100.0 mg/g); 1-heptacosanol (C27H55OH, 1.0–30.0 mg/g); 1-octacosanol (C28H57OH, 600.0–700.0 mg/g); 1-nonacosanol (C29H59OH, 1.0–20.0 mg/g); 1-triacontanol (C30H61OH, 100.0–150.0); 1-dotriacontanol (C32H65OH, 50.0–100.0 mg/g); and 1-tetratriacontanol (C34H69OH, 1.0–50.0 mg/g). Briefly, the participants were stratified into the policosanol or placebo groups and took two tablets containing policosanol 10 mg (Raydel^®) or a placebo per day. The tablet in the policosanol group included policosanol (10 mg), hydroxypropyl cellulose, carboxymethyl cellulose, maltodextrin, lactose, and crystalline cellulose. In the placebo group, the tablet contained maltodextrin (10 mg), rather than policosanol. The Raydel^® policosanol tablet was acquired from Raydel Japan (Tokyo, Japan), which was manufactured with Cuban policosanol at Raydel Australia (Thornleigh, Sydney). According to the exclusion criteria described below, the placebo group comprised 17 participants (male = 9, female = 8), whereas the policosanol group included 15 participants (male = 8, female = 7).

In the present study, the inclusion criteria were the same as those described in the main randomized clinical trial (15). All participants were advised to refrain from ingesting excess food/cholesterol and alcohol drinking. Moreover, before the study and during the study period, smoking both directly and indirectly was not allowed. The inclusion criteria were as follows: age between 20 and 65 years and serum LDL-C levels in the normal and mildly elevated range (120–159 mg/dL). In the current study, the exclusion criteria were more strict in terms of dietary habits, including food and drink, in each self-questionnaire to eliminate the impact on lipid profile, as referenced from the Japan Atherosclerosis Society Guidelines for the Prevention of Atherosclerotic Cardiovascular Diseases 2017 (25). The exclusion criteria were as follows: (1) maintenance treatment for metabolic disorders, including dyslipidemia, hypertension, and diabetes; (2) severe hepatic, renal, cardiac, respiratory, endocrinological, and metabolic diseases; (3) allergies; (4) heavy drinkers, consuming >30 g and 15 g of alcohol per day for men and women, respectively; (5) taking medicine or functional food products that may affect lipid metabolism, including an increase in HDL-C concentration or a decrease in LDL-C concentration and a decrease in the triglyceride concentration; (6) current and past smokers; (7) pregnant or lactating women or women planning to become pregnant during the study period; (8) who donated >200 mL of blood within 1 month or 400 mL of blood within 3 months prior to clinical trial initiation; (9) who participated in other clinical trials within the last 3 months or is currently participating in another clinical trial; (10) those who consumed >2,000 kcal per day or cholesterol intake >600 mg per day; and (11) others considered unsuitable for the study as per the principal investigator’s discretion. The ethics committee of the Koseikai Fukuda Internal Medicine Clinic reviewed and approved the study protocol (Osaka, Japan, IRB approval number 15000074, approval date on September 18, 2021), which was conducted in accordance to the ethical guidelines stipulated in the 1975 Declaration of Helsinki. The protocol was also registered with the University Hospital Medical Information Network Clinical Trial Registry (UMIN000046345). All participants provided written informed consent prior to initiation of any study procedure.

According to the methods described in the randomized clinical study, anthropometric measurement and blood samples were collected from the participants (15). The samples were analyzed by BML Inc. (Tokyo, Japan) for lipid, glucose, and inflammatory factors. According to standard protocols, serum lipoproteins were isolated via ultracentrifugation (26), which were then divided as follows: very-low-density lipoprotein (VLDL) (d < 1.019 g/mL), LDL (1.019 < d < 1.063), HDL₂ (1.063 < d < 1.125), and HDL₃ (1.125 < d < 1.225). The total cholesterol and triglyceride levels in each lipoprotein were measured using commercially available kits (Wako Pure Chemical, Osaka, Japan) after lipoprotein purification. Lipoprotein characterization, including the shape and size of the HDL and its particle number, was conducted via transmission electron microscopy (Hitachi H-7800; Ibaraki, Japan) at 40,000× magnification, as described in the previous report (27). Briefly, the HDL size was measured using Image J software version 1.53r¹ and Hitachi EMIP-EX software (Ver. 07.13, Hitachi Tokyo, Japan) with approximately 70 randomly selected particles. To measure the particle size, manually and randomly selected individual particle image using a tablet pen (CLT-6100WL, Wacom, Saitama, Japan) were calculated using the software. HDL₂ particle size and diameter (10–15 nm) were larger than that of HDL₃ (7–9 nm of diameter) using Hitachi EMIP-EX software (Ver. 07.13, Hitachi Tokyo, Japan).

The lipoprotein subclass and composition in plasma, such as chylomicron, VLDL, LDL, and HDL, were analyzed through gel permeation high-performance liquid chromatography (HPLC) using LipoSEARCH (Skylight Biotech, Inc., Akita, Japan) (28, 29). In the HPLC analysis, lipoproteins can be stratified into 20 lipoprotein subclass groups, which are as follows: chylomicron (fractions 1–2), VLDL (fractions 3–7: large, 3–5; medium, 6; small, 7), LDL (fractions 8–13: large, 8; medium, 9; small, 10–13), and HDL (fractions 14–20: large, 14–16; medium, 17; small, 18–20). After the analysis, the cholesterol and triglyceride contents in those subclasses were quantified.

J774A.1 cells (JCRB cell bank, Osaka, Japan) were purchased and cultured in Dulbecco’s Modified Eagle Medium with 4.5 g/L of a high glucose (Sigma-Aldrich, St Louis, MO, United States) medium containing 10% fetal bovine serum (Life Technologies Co, Carlsbad, CA, United States) and 0.5% penicillin/streptomycin.

22(R)-hydroxycholesterol and 9-cis-retinoic acid were purchased from Sigma-Aldrich and LKT Laboratories, Inc. (St Paul, MN, United States), respectively, and were dissolved using a dimethyl sulfoxide solution. Fatty-acid-free bovine serum albumin (BSA) was purchased from Calbiochem (Merck KGaA, Darmstadt, Germany).

The cellular CEC was measured as described previously (30–34). Briefly, J774A.1 cells were radiolabeled with 5 uCi/mL of [1,2-3H] cholesterol (PerkinElmer, Waltham, MA, United States) for 24 h. J774A.1 cells were treated with 5 μmol/L 22(R)-hydroxycholesterol, an LXR agonist, and 2.5 μmol/L 9-cis-retinoic acid, an RXR agonist to activate ABCA1 and ABCG1 for 18 h. Then, 2.0% apolipoprotein B-depleted plasma, which included HDL through ApoB-depleted treatment with the phosphotungstic acid-Mg precipitation method, was added to the cells for 4 h to extract cholesterol from the cells to the supernatant. Finally, the liquid scintillation count was measured, and the efflux rates of 3H-cholesterol from the cells to the medium were analyzed. Serum-free medium containing 0.2% BSA was used as the basis solution for radiolabeled treatment, stimulation, and plasma addition for all experiments on the HDL CEC.

Data were expressed as the mean ± standard deviation (SD) or ± standard error of mean, and median with interquartile range as appropriate. The Kolmogorov–Smirnov test for normality was used to evaluate the baseline population characteristics, and the difference was analyzed using an unpaired t-test with normality or Mann–Whitney U test without normality. For triglycerides, apolipoprotein B-48 (ApoB-48), and high sensitivity C-reactive protein (hsCRP), log-transformed values, which were normally distributed, were used to compare them. Using a homogeneity test of the variances through Levene’s statistics for normality, HDL₂ and HDL₃ evaluation was performed. Then, a paired t-test was performed with normality, and if without normality, nonparametric statistics were performed using the Kruskal–Wallis test, for HDL₂ and HDL₃ morphology and composition analysis.

For lipoprotein fractions after normality evaluation, the HPLC data difference was analyzed using an unpaired t-test with normality or by Mann–Whitney U test with log-transformed values without normality. In a four-group comparison categorized according to baseline HDL-C levels and sex, differences between groups were analyzed using the Kruskal–Wallis test with Dunn’s multiple comparison test. Using a simple linear regression with Pearson’s product–moment correlation co-efficient with normality or Spearman’s rank correlation coefficient without normality, a single correlation analysis was evaluated. Statistical significance was set at a p value < 0.05.

The GraphPad Prism version 8 software (GraphPad software LLC., San Diego, CA, United States) and the JMP Version 12.0 software package (SAS Institute Inc., NC, United States) were used for the statistical analyses, except in the HDL₂ and HDL₃ evaluations. In the HDL₂ and HDL₃ evaluations, statistical power was estimated using the program G*Power 3.1.9.7 (G*Power from University of Düsseldorf, Düsseldorf, Germany). SPSS software version 29.0 (IBM, Chicago, IL, USA) was used to analyze data.

In this paper, lipid profiles and function were analyzed using data from previously published randomized clinical trials [placebo (n = 17) or policosanol (20 mg/day, n = 15), 12 weeks] (15) to examine the effects and mechanisms of policosanol on lipids. The policosanol effects were investigated through observation of the changes from baseline (week 0) to the end (week 12) of the study in Japanese healthy participants. At baseline, no difference in any factor was found between the placebo and policosanol groups (Table 1).

The policosanol effects after a 12-week placebo or policosanol supplementation are shown in Figure 1. The HDL CEC upregulation was markedly increased in the policosanol group at week 12, as compared with the placebo group (Figure 1A). Similarly, the HDL-C concentration changes were significantly higher in the policosanol group (Figure 1B). The ApoA-I (Figure 1C) and ApoA-II (Figure 1D) level changes, which are two major HDL constituent proteins, were also significantly increased in the policosanol group at week 12.

Conversely, changes in the total cholesterol (Figure 2A), LDL-C (Figure 2B), and triglyceride (Figure 2C) levels from baseline were not different between the placebo and policosanol groups at week 12. No difference in the ApoB-100 (Figure 2D) and ApoB-48 (Figure 2E) levels, located on the surface of VLDL/LDL and chylomicron, respectively, was also found between the placebo and policosanol groups. At week 12, glucose-related factor levels such as HbA1c (Figure 2F) and blood glucose (Figure 2G) and the inflammatory factor hsCRP (Figure 2H) did not differ between the two groups.

Next, HDL form and composition alterations induced by policosanol were evaluated as policosanol particularly affected HDL. After lipoprotein isolation using the ultracentrifugation method, transmission electron microscopy revealed that the HDL particle number in the policosanol group at week 12 was higher, with more distinct particle morphology than that at week 0 (Figure 3A). Contrarily, the placebo group failed to show any notable change in particle morphology between weeks 0 and 12. In the policosanol group, the HDL₂ particle size at week 12 was 22% higher than that of week 0 (p < 0.001). The placebo group at week 12 revealed a 10% decrease in particle size at week 12 than at week 0 (p = 0.020) with a more aggregated pattern than that of the policosanol group at week 12 (Figures 3A,B). No remarkable difference in the cholesterol and triglyceride levels in the HDL₂ composition was noted in the policosanol in Table 2. Additionally, fluorescence intensity (FI) indicated glycation extent of yellowish fluorescence. Because the lower FI of HDL in the policosanol group was shown, it indicated that policosanol modulated a decreased extent of glycation in HDL.

In Figure 3C, transmission electron microscopy revealed that at week 12, the HDL₃ particle size in the policosanol group was 1.4-fold higher than that at week 0 (p < 0.001), with a more distinct particle shape. The placebo group failed to show a notable change in HDL₃ particle morphology. In Table 2, the policosanol group showed almost no change, even a slight increase in the cholesterol content in HDL₃ between weeks 0 and 12, whereas the placebo group revealed a 31% decrease in HDL₃ between weeks 0 and 12. Interestingly, at week 12, the triglyceride content in the policosanol group was 25% lower than that at week 0, whereas the placebo group demonstrated no notable change between weeks 0 and 12. These results indicate that policosanol supplementation also affects HDL₃ size enlargement, whereas the placebo group revealed no difference in particle size.

Furthermore, to verify the cholesterol and triglyceride contents in lipoproteins defined by particle size in detail, HPLC analysis was performed (28, 29). At baseline, 20 lipoprotein fractions from a chylomicron to HDL are shown in Table 3. The difference at baseline was only observed in the cholesterol content of VLDL (fraction 5) and triglyceride content of HDL (fraction 17). However, it was deemed to be unimportant because the values were of very low proportion compared to the total amount in both cholesterol and triglyceride.

The alterations in cholesterol content in each HDL subclass (largest to smallest HDL particles with seven fractions) were shown in Figure 4. The HPLC analysis revealed that policosanol increased the cholesterol content in the largest to medium HDL particles (four fractions) (Figures 4A–D), except in small HDL particles (Figures 4E–G), compared with the placebo. As shown in Figure 5, triglyceride content changes from baseline was reduced in the policosanol group in terms of the medium to small HDL particles (17–18 fractions); however, no change was noted in other HDL fractions. These microscopic and HPLC outcomes revealed that policosanol induced the growth of larger HDL particles, increased the cholesterol content of larger HDL particles, and reduced the triglyceride content of smaller HDL particles.

In terms of the changes in other lipoproteins induced by policosanol supplementation, cholesterol and triglyceride in the chylomicron (fractions 1–2), VLDL (fractions 3–7), and LDL (fractions 8–13) were slightly different at week 12 (Supplementary Figure S1). Cholesterol content in the medium-sized VLDL (fraction 6, Supplementary Figure S1F) and smallest LDL (fraction 13, Supplementary Figure S1M) was decreased and increased, respectively. However, the difference was believed to be nonsignificant because (1) there were no remarkable alterations in the total LDL-C levels (Figure 2B), (2) the cholesterol levels in two fractions were lower compared with the total cholesterol levels, and (3) same difference was not observed in the consecutive fractions. As shown in Supplementary Figure S2, the triglyceride content in large to medium to small VLDL (fractions 5–7) was decreased (Supplementary Figures S2E–G) in the policosanol group, as compared with the placebo.

We further examined whether HDL-C levels at baseline were associated with policosanol effects, such as the changes in the HDL CEC and cholesterol/component proteins in HDL. In Table 4, data of four groups stratified by baseline (week 0) HDL-C levels (placebo with low HDL-C levels, n = 8; placebo with high HDL-C levels, n = 9; policosanol with low HDL-C levels, n = 8; policosanol with high HDL-C levels, n = 7) are presented.

Comparing the four groups at week 12, HDL CEC was upregulated in only the high-HDL-C group with policosanol, as compared with the other three groups (Figure 6A). Likewise, HDL-C (Figure 6B) and ApoA-I (Figure 6C) levels were elevated in only the high HDL-C group of policosanol supplementation. However, policosanol did not change the ApoA-II levels in the four groups stratified by HDL-C levels at week 0 (Figure 6D).

Similarly, the sex-specific effects of policosanol supplementation were explored in four groups, as shown in Supplementary Table S1 (males in the placebo group, n = 9; females in the placebo group, n = 8; males in the policosanol group, n = 8, females in the policosanol group, n = 7). No difference between the four groups was noted at baseline. Surprisingly, policosanol supplementation increased the HDL CEC and ApoA-I levels in only females with policosanol and HDL-C levels in both sexes with policosanol compared to that in placebo (Supplementary Figures S3A–C). However, ApoA-II levels were not altered in four groups (Supplementary Figure S3D).

Finally, to determine whether the values were related to the alteration of other HDL/LDL-related factors in all healthy participants, changes in HDL CEC from week 0 to week 12 were examined. The HDL CEC alteration was associated with changes in HDL-C (Figure 7A), ApoA-I (Figure 7B), and ApoA-II (Figure 7C) levels, but not in ApoB-100 levels (Figure 7D).