Uptake of Vitamins D2, D3, D4, D5, D6, and D7 Solubilized in Mixed Micelles by Human Intestinal Cells, Caco-2, an Enhancing Effect of Lysophosphatidylcholine on the Cellular Uptake, and Estimation of Vitamins D' Biological Activities

Abstract

Vitamins D have various biological activities, as well as intestinal calcium absorption. There has been recent concern about insufficient vitamin D intake. In addition to vitamins D₂ and D₃, there are lesser-known vitamins D₄-D₇. We synthesized vitamins D₅-D₇, which are not commercially available, and then evaluated and compared the mixed micelles-solubilized vitamins D uptake by Caco-2 cells. Except for vitamin D₅, the uptake amounts of vitamins D₄-D₇ by differentiated Caco-2 cells were similar to those of vitamins D₂ and D₃. The facilitative diffusion rate in the ezetimibe inhibited pathway was approximately 20% for each vitamin D type, suggesting that they would pass through the pathway at a similar rate. Lysophosphatidylcholine enhanced each vitamin D uptake by approximately 2.5-fold. Lysophosphatidylcholine showed an enhancing effect on vitamin D uptake by reducing the intercellular barrier formation of Caco-2 cells by reducing cellular cholesterol, suggesting that increasing the uptakes of vitamins D and/or co-ingesting them with lysophosphatidylcholine, would improve vitamin D insufficiency. The various biological activities in the activated form of vitamins D₄-D₇ were estimated by Prediction of Activity Spectra for Substances (PASS) online simulation. These may have some biological activities, supporting the potential as nutritional components.

Keywords: Caco-2 cells; PASS; intercellular barrier formation; intestinal uptake; lysophosphatidylcholine; mixed micelles; vitamin D.

Figure 1

Chemical structures of vitamins D. The numbers in the structural formula indicate the locations of the carbons.

Figure 2

Uptake of vitamin D solubilized in mixed micelles by differentiated Caco-2 cells and the effects of lysophosphatidylcholine. Differentiated Caco-2 cells were incubated for 2 h with vitamin D in mixed micelles without lysophosphatidylcholine and mixed micelles with lysophosphatidylcholine, called NoPC mixed micelles (open bars) and lysoPC mixed micelles (filled bars), respectively. (a) Vitamin D uptake by differentiated Caco-2 cells. (b) Residual concentrations of vitamin D in the mixed micelles (micellar vitamin D) after 2-h incubation with the cells. Data are presented as the means ± standard deviation of four wells in a single experiment. Replicate experiments showed a similar trend. Statistical analyses were performed between NoPC mixed micelle and lysoPC mixed micelle in same vitamin D, among 6 kinds of vitamins D in NoPC mixed micelles, and among 6 kinds of vitamins D in lysoPC mixed micelles in each graph. The asterisk indicates a value with significant difference as determined by unpaired t-test (p < 0.05). Values not sharing a common letter were significantly different as determined by Tukey–Kramer test (p < 0.05).

Figure 3

Inhibitory effects of ezetimibe on the uptake of vitamin D solubilized in mixed micelles by differentiated Caco-2 cells. Differentiated Caco-2 cells were pretreated for 2 h with the vehicle (1.5% dimethyl sulfoxide (DMSO), open bars) alone or with 150 μM ezetimibe (filled bars), which is an inhibitor of the protein Niemann-Pick C1-like 1 (NPC1L1), and then incubated for 2 h with vitamin D in NoPC mixed micelles (left side) and lysoPC mixed micelles (right side). (a,b): Vitamin D uptake by differentiated Caco-2 cells. (c,d): Residual concentrations of micellar vitamin D after 2-h incubation with the cells. Data are presented as the means ± standard deviation of four wells in a single experiment. Replicate experiments showed a similar trend. Statistical analysis was performed to compare each vitamin D with and without ezetimibe. The asterisk indicates a value with significant difference as determined by unpaired t-test (p < 0.05).

Figure 4

Uptake of vitamin D solubilized in mixed micelles by dispersed Caco-2 cells. Dispersed Caco-2 cells without cell-cell adhesion/cell-matrix adhesion were incubated for 2 h with vitamin D in NoPC mixed micelles. The cells were prepared by dispersing the cells with trypsin. (a) Vitamin D uptake by dispersed Caco-2 cells. (b) Residual concentrations of the micellar vitamin D after 2-h incubation with the cells. Data are presented as the means ± standard deviation of four wells in a single experiment. Replicate experiments showed a similar trend. Statistical analysis was performed among all six samples in each graph. Values not sharing a common letter were significantly different as determined by Tukey–Kramer test (p < 0.05).

Figure 5

Effects of lysophosphatidylcholine on the uptake of vitamin D₂ solubilized in mixed micelles by dispersed Caco-2 cells. Dispersed Caco-2 cells without cell-cell adhesion/cell-matrix adhesion were incubated for 2 h with vitamin D₂ in NoPC mixed micelles and lysoPC mixed micelles. (a) Vitamin-D₂ uptake by dispersed Caco-2 cells. (b) Residual concentrations of micellar vitamin D₂ after 2-h incubation with the cells. Data are presented as the means ± standard deviation of four wells in a single experiment. Replicate experiments showed a similar trend. Statistical analysis was performed among all six samples in each graph. The asterisks indicate significant differences as determined by unpaired t-test (p < 0.05).

Figure 6

Effects of lysophosphatidylcholine on the uptake of vitamin D₂ by Caco-2 cells with insufficient cell-cell adhesion. Caco-2 cells with insufficient cell-cell adhesion/cell-matrix adhesion were prepared by cultivating for 3, 5, and 7 d after seeding in the culture plate. (a) These cells were observed by phase-contrast microscopy (100×). The white line in the photo of 7 d indicates the scale bar (100 μm). The adhered Caco-2 cells were then incubated for 2 h with vitamin D₂ in NoPC mixed micelles (circles) and lysoPC mixed micelles (triangles). (b) Vitamin-D₂ uptake by the adherent Caco-2 cells. (c) Residual concentrations of the micellar vitamin D₂ after 2-h incubation with the cells. Data are presented as the means ± standard deviation of four wells in a single experiment. Replicate experiments showed a similar trend. Statistical analysis was performed between two mixed micelles at the same cultivation time (3, 5, and 7 d). The asterisk indicates a value with significant difference as determined by unpaired t-test (p < 0.05).

Figure 7

Reducing effect of lysophosphatidylcholine in mixed micelles on cellular cholesterol amounts in the differentiated Caco-2 cells. The differentiated Caco-2 cells were incubated for 2 h with the mixed micelles containing lysophosphatidylcholine at 0–250 μM. The mixed micelles contained no vitamin D. Data are presented as the means ± standard deviation of four wells in a single experiment. Replicate experiments showed a similar trend. The asterisks indicate significant differences from the value of lysophosphatidylcholine at 0 μM as determined by Dunnett’s test (p < 0.05).