Subject recruitment
The individuals (n = 38: female 31 and male 7) aged 38–77 years were recruited in the Dyslipidemia Outpatient Unit of the Endocrinology and Metabolism Service of the Clinical Hospital of the University of Sao Paulo, Brazil; members of the staff of the University of Sao Paulo, Brazil were also included. The participants were invited for screening of body weight and height; blood samples were collected for lipids profile determination. The inclusion criteria were: body mass index (BMI) between 20 and 30 kg/m2; total cholesterol between 200–300 mg/dL, LDL-C concentrations ≥ 130 mg/dL, and triglycerides ≤ 250 mg/dL (Table 1). Exclusion criteria were: use of lipid-lowering medication or a prescribed diet in the last month; alcohol abuse or illicit drug users; pregnancy or breast feeding; smoking; diabetes mellitus, hypothyroidism, renal or hepatic diseases or participation in another lifestyle or pharmaceutical intervention studies. All subjects provided informed written consent. The Ethics in Research Committee of the Hospital of the University of Sao Paulo Medical School approved the study protocol (CAPPesq n° 112/06).
Table 1
Subjects characteristics at baseline
Parameter | Mean ± SD |
n | 38 |
Age (years) | 58 ± 12 |
Weight (Kg) | 64 ± 10 |
BMI (kg/m2) | 25.3 ± 2.4 |
Total cholesterol (mg/dL) | 245 ± 34 |
Triglycerides (mg/dL) | 141 ± 53 |
LDL-C (mg/dL) | 165 ± 34 |
HDL-C (mg/dL) | 49 ± 12 |
BMI, body mass index; LDL-C, low-density lipoprotein cholesterol; HDL-C: high density lipoprotein cholesterol |
Study Design
The present study was a randomized, double-blind, placebo-controlled dietary intervention trial with each study period lasting 4 weeks. Initially, all the participants were submitted to a 3-week run-in period in which they received the placebo product (soy milk) to test adherence to the protocol. After baseline period the individuals were randomly assigned to placebo or to phytosterol groups for 4 weeks; after that a reverse sequence was immediately carried out. Placebo group received 400 mL of soy milk daily; phytosterol group received 400 mL of soy milk enriched with 1.6 g of PS, as follow: 78% β-sitosterol-ester, 13% sitostanol-ester, 5.3% campesterol-ester and 0.5% campestanol-ester (Table 2). Blood samples were drawn for biochemical analysis from fasting participants on the last day of each period study. All participants were advised to maintain body weight and follow a normocaloric diet based on the NCEP-ATPIII recommendation [8]: 30% of energy as fat, < 10% of energy as saturated fat, and < 300 mg cholesterol/day and was recommended not to consume products enriched with phytosterol during the study. Nutritional monitoring was carried out by a registered dietitian using a 24-hour dietary recall to ensure adherence to the prescribed diet and also to estimate the food intake. Soy milk was weekly supplied at the same day of the body weight measurement; patients were instructed to consume the soy milk or PS-enriched soy milk twice daily, at lunch and dinner.
Table 2
Soy milk nutritional composition per portion (200 mL)*.
Nutritional composition | Soy milk | Soy milk + PS |
Energy (kcal) | 138 | 144 |
Protein (g) | 6.5 | 6.5 |
Total fat (g) | 4.4 | 5.0 |
Polyunsaturated fat | 2.3 | 2.5 |
Monounsaturated fat | 1.0 | 1.1 |
Saturated fat | 0.7 | 0.9 |
Trans fatty acid | 0 | 0 |
Cholesterol (mg) | 0 | 0 |
Carbohydrates (g) | 18.2 | 18.2 |
Total sugar | 14.1 | 14.1 |
Lactose | 0 | 0 |
Phytosterol (g) | 0 | 0.8 |
β-sitosterol-ester | | 0.63 |
Sitostanol-ester | | 0.10 |
Campesterol-ester | | 0.05 |
Campestanol-ester | | 0.005 |
Sodium (g) | 0.1 | 0.1 |
*Provided by Nestle Company, São Paulo, Brazil |
Blood Sampling
After fasting for 12 hours, blood samples were collected into tubes containing Ethylenediamine tetraacetic acid (EDTA). Plasma was immediately separated by centrifugation (1300 g, 15 min, 4 °C; RT6000B; Sorvall Instruments, DuPont Co, Newton, CT), and the following preservatives were added: 0.25% chloramphenicol plus 0.5% gentamycin (20 µL/mL), 2 mmol benzamidine/L (5 µL/mL), 10 mmol phenyl-methyl-sulfonyl fluoride/L (0.5 µL/mL), and aprotinin (0.5 µL/mL). For HDL-C measurements, plasma was mixed and kept at room temperature for 10 min and then centrifuged (700 g, 30 min, 4ºC). Aliquots (500 µL) were stored at -70ºC.Total plasma and serum were stored at -70ºC. All measurements were performed in duplicate at the end of the study. All samples from one subject were analyzed within the same analytical run.
Serum Sterols Analyses
Plasma precursors of cholesterol synthesis (desmosterol, lathosterol) and phytosterols (campesterol and sitosterol) were measured in samples (100 µL) added 5α-cholestane (1 µg) as the internal standard, hydrolyzed with KOH in ethanol (1 mol/l, 1 ml) at 60 °C (1 h) and extracted with hexane. Sterols were derivatized with a sylilating solution (pyridine and BSTFA (N,O-bis (trimethylsilyl) trifluoroacetamide) + 1% TMCS (trimethylchlorosilane) (1:1, v/v) (Supelco 33155-U) for 1 h at 60 °C [28]. The quantification was performed comparing the peak areas of the standard curve and corrected for internal standards. Plasma non-cholesterol sterols (µg) were expressed as ratio of plasma total cholesterol (mg).
Statistical analysis
Comparisons between the placebo and phytosterol groups were analysed by paired Student’s t test. The influence of degree of hypercholesterolemia over the PS response and PS response patterns related to LDL-C were analysed by unpaired Student’s t test. Data are shown as means and standard deviation. The analyses were performed utilizing the GraphPad Prisma version 4.00 and significance level considered as p < 0.05.