Supplementation of test meals with fat-free phytosterol products can reduce cholesterol micellarization during simulated digestion and cholesterol accumulation by Caco-2 cells

Phytosterols have been shown to reduce cholesterol absorption in humans. Supplementing phytosterols in fat-free formulations, however, has yielded controversial results. In the present study, we investigated the effect of supplementing test meals with different fat-free phytosterol products on cholesterol incorporation into mixed micelles during simulated digestion and accumulation of micellar cholesterol by Caco-2 cells: control orange juice (OJ), orange juice supplemented with either multivitamin/multimineral tablets (MVT), multivitamin/multimineral tablets containing phytosterols (MVT+P), and phytosterol powder (PP). These combinations were added to Ensure-based test meals and spiked with cholesterol of natural isotopic composition or ¹³C₂-cholesterol to differentiate external from endogenous cholesterol. After simulated gastric/small intestinal digestion, micelle fractions were analyzed for cholesterol enzymatically (n = 6-20/product) and by high-performance liquid chromatography-tandem mass spectrometry (n = 12/product) and added to Caco-2 cells to determine the accumulation of ¹³C ₂-cholesterol (n = 10-24/product). As compared to OJ, PP and MVT+P significantly decreased cholesterol micellarization (determined enzymatically) by 70 ± 39 (mean ± SD) and 70 ± 39%, respectively (P < 0.001, Bonferroni). The stable isotope experiments revealed that both PP and MVT+P reduced cholesterol micellarization [by 25 ±12 (P = 0.055) and 21 ± 8% (P = 0.020), respectively, Fisher's protected LSD test] and Caco-2 cell accumulation (by 28 ± 8 and 10 ±8%, respectively; P < 0.010, Bonferroni). OJ+P did not inhibit micellarization or accumulation of cholesterol by Caco-2 cells. This study shows that fat-free phytosterol- containing products can significantly inhibit cholesterol micellarization and Caco-2 cell bioaccessibility, albeit to different extents depending on individual formulations. This is most likely explained by inhibition of cholesterol micellarization.