*2.5. Statistical Analysis*

The study population was divided into three groups according to the tercile distributions of plant sterol intakes (separately for total and individual phytosterols). All analyses were performed according to gender and overall. Quantitative variables were presented as mean (standard deviation) and/or median (interquartile range), while qualitative variables were presented as percentages. Mean values of plant sterol intake with a 95% confidence interval (95% CI), adjusted for age, were calculated using the general linear model and the Tukey-Kramer test was chosen for multiple comparisons, if appropriate. The odds ratios (ORs) with 95% CI for CVD were evaluated using logistic regression analysis in relation to total and particular phytosterol intake. Two models were applied: model 1, unadjusted in men and women but adjusted for sex, and combined, and model 2, adjusted for age, consumption of lipid-lowering drugs, HDI, BMI, alcohol intake, and, additionally, for sex, for the entire population. The first tercile (T1) in each model was adopted as a reference. Statistical analyses were carried out using SAS software version 9.4 (SAS Institute Inc., Cary, NC, USA). A *p*-value less than 0.05 was considered statistically significant.

#### **3. Results**

The general characteristics of the study participants are shown in Table 1. The mean age of the entire study group was 49.58 years. The highest percentage of the study participants had hypercholesterolemia 67.3% and hypertension 45.22%. CVD was diagnosed in 20% of the studied population, while diabetes in 10.82%.

Table 2 shows the phytosterol intake according to age, presence of diabetes, and CVD. The results are presented for men, women, and the entire study group. Dietary phytosterol content was found to be age-dependent and generally highest among the youngest age group and lowest among those aged 65 years and older. Among men and in the entire study group, sterol intake was significantly lower among people with diabetes (results were adjusted for age). No significant differences were found for women. With respect to CVD, plant sterol intake was lower among both men and women with CVD (*p* = 0.0016) and for both genders (*p* < 0.0001). With regard to diabetes, such a relationship was observed for men and the entire group, but not for women. With respect to individual plant sterols, we found that dietary intake of phytosterols was lower among both men with CVD and women and among men with diabetes (except campesterol in men with diabetes). No differences were found between women with diabetes and healthy women. For the whole group, only campesterol was not statistically significant. The intake of individual plant sterols with the fortified margarine was not considered, because manufacturers only reported the total phytosterol content. Thus, it was not possible to determine what the individual plant sterol content of the margarine was.

Table 3 shows terciles of plant sterol intake with food, and with food including phytosterol-enriched margarine. Terciles of individual plant sterol intake for the entire study group and by gender were used as means (crude, adjusted), medians, and ranges for particular phytosterols intake. Among men, the median plant sterol intake in the first tercile was 183, in the second tercile 292, and in the third tercile 456 mg/d. For food intake, including margarine with phytosterols, the values were 184; 294, and 459 mg/d, respectively. Among women, the median intakes of plant sterols with diet were: 146 in the first tercile, 231 in the second tercile, and 350 mg/d in the third tercile. For food intake, including margarine with phytosterols, these values for women were, respectively: 147; 232, and 352 mg/d. For individual plant sterols, they are ranked in Table 3 by the volume of intake. Among both men and women, beta-sitosterol intake predominated, with a median range of 112–280 mg/d per tercile among men and 91–222 mg/d among women. For campesterol, the median range was 31–107 mg/d among men and 24–78 mg/d among women, and for stigmasterol, 12–39 mg/d among men and 12–34 mg/d for women.

The odds ratio of developing CVD was related to phytosterol intake with diet (Figure 1). In the crude model, it was found that in both men and women, and in the entire study group (adjusted for gender), OR of CVD were significantly lower in the second and third terciles compared to the first terciles, with the lowest incidence of CVD in the third tercile. After adjusting for confounding factors, among men statistical significance was maintained, except for the second tercile of beta-sitosterol intake. Among women, only the intake of total plant sterols from the diet and their total intake together with margarine in the third tercile, and the intake of beta-sitosterol in the third tercile remained statistically significant. In the entire study group, significant values were observed in the third tercile of total plant sterol intake (without and with phytosterol-enriched products), and for all individual plant sterols.


**Table 1.** General characteristics of the studied population.

<sup>1</sup> Hypertension: systolic blood pressure SBP ≥ 140 mmHg or diastolic blood pressure DBP ≥ 90 mmHg, or use of antihypertensive drugs. <sup>2</sup> Hypercholesterolemia: total cholesterol ≥ 5 mmol/L or LDL cholesterol ≥ 3 mmol/L or the participant was taking lipid-lowering medication. <sup>3</sup> Diabetes: blood glucose level was ≥7.0 mmol/L or diabetes was declared in an interview.


**Table 2.** Phytosterol intakes depending on age, diabetes, and CVD.

Results adjusted for age in men and women and additionally for sex in total; adjustment not applicable to the age groups.


**Table 3.** Intake of

phytosterols

 in terciles. \* Results were adjusted for age in men and women and additionally for sex in total.


**Figure 1.** Odds ratio (95% confidence interval) for CVD in relation to total and individual phytosterol intake (relative to 1st tercile). T2—2nd tercile; T3—3rd tercile; OR—odds ratio; AOR—adjusted odds ratio; ORs were unadjusted in men and women but adjusted for sex combined; AORs—adjusted for age, lipid-lowering medication, HDI, BMI, alcohol, and additionally for sex in total.

We also investigated whether the results obtained could be biased by diet (Table 4). For this purpose, data from the extreme terciles of total and single plant sterol intake before and after energy adjustment were presented by sex and the entire study group. It was found that both before and after adjustment the results were significant for total and single dietary plant sterol. For intakes of phytosterol-enriched margarine, a significant difference was found between the first and third terciles before energy adjustment, which did not occur after adjustment. Intakes of polyphenols, antioxidants, dietary fiber, and HDI were also divided according to the tercile of total and individual plant sterol intake, with polyphenols, antioxidants, and dietary fiber adjusted for energy value. It was found that before adjustment, dietary polyphenol, antioxidant, and fiber contents were higher in the third tercile among both men and women and in the group as a whole (*p* < 0.0001). After adjustment for energy, differences were not observed. Diet quality, as measured by HDI, was significantly higher in the third tercile of plant sterol intake for both men and women and for the entire study group (*p* < 0.0001).

Intakes of atherogenic and antiatherogenic products were also examined in the first and third terciles of total and individual phytosterol intake (Table 5). For atherogenic products, butter and animal fat consumption was found to be higher in the third tercile of plant sterol intake, but after adjustment for energy there was an inverse difference, i.e., with higher plant sterol intake, animal fat and butter consumption was lower. For red meat and meat products before and after adjustment for energy, consumption was higher in the third tercile. All the above observations were true for both men and women and for the entire study group.


**Table 4.** Dietary quality in relation to tercile of dietary total and individual phytosterol intake (1st tercile vs.

 3rd tercile).

Results were adjusted for age in men and women and additionally for gender in men and women overall.


**Table 5.** Consumption of selected products by tercile of phytosterol intake (1st tercile vs. 3rd

 tercile).

In the case of intake of antiatherogenic products, it was found that in both sexes and in the entire study group, both before and after adjustment for energy, the intake of vegetable oils, vegetable fats, fish, fruits, legumes, nuts, and seeds was higher in the third tercile of plant sterol intake. For soft margarine and vegetables, there were similar differences among men and the overall study group, but not among women. In women, after adjustment, differences were not observed. For whole grain bread, higher consumption by both sexes and in the entire study group was observed in the third tercile, but after adjusting for energy, differences were not significant.
