In vitro study of triglyceride lipolysis and phase distribution of the reaction products and cholesterol: effects of calcium and bicarbonate

We describe a relatively simple in vitro model for triglyceride (TG) lipolysis which mimics closely the conditions in the human stomach and small intestine. The main model advantages are: (1) as in vivo, sodium bicarbonate is used for buffering; (2) the pH-profile in the small intestine is closely matched; (3) the experimental procedure does not include complex equipment. To test its performance, the proposed in vitro model is applied to quantify the effects of Ca2+, pH, and bicarbonate on the degree of TG lipolysis and on the solubilization of the lipolysis products and cholesterol in the aqueous phase. We found that TG lipolysis passes through a shallow minimum at 3.5 mM Ca2+ when varying the calcium concentration between 1 and 11 mM, while the presence of bicarbonate and the increase of pH led to a higher degree of lipolysis. Centrifugation and filtration were used to separate the aqueous phase and to study the solubilisation of the lipophilic components in the aqueous phase. We found that the solubilized cholesterol increases linearly with the concentration of free fatty acids (FFA) which is evidence for co-solubilization of these two components in the bile micelles. At high Ca2+ concentrations, aggregates larger than 300 nm were observed by cryo-microscopy and light scattering, which solubilize well cholesterol and saturated FFA. In contrast, the monoglycerides were always predominantly solubilized in the small bile micelles with diameters around 4 nm.