Phospholipids c test

Encapsulation efficiency of P-KH liposome was evaluated by using a model drug (calcein). P-KH liposome was prepared with 10 mM calcein solution. Nonencapsulated calcein was removed from P-KH liposome suspension by gel filtration (Sepharose 4B; GE Healthcare, Little Chalfont, UK). Concentration of DMPC was measured by choline oxidase/DAOS method (Phospholipids C Test Wako). Fluorescence intensity of calcein in the P-KH liposome was measured by fluorescence spectrophotometer FP-6500 (Jasco, Tokyo, Japan), after disruption of P-KH liposome by Triton X-100 in order to evaluate strictly encapsulation efficiency without effect of calcein self-quenching [19 (link)]. The number of entrapped calcein molecules in a liposome was calculated as follows: $\begin{array}{c}\frac{\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\hspace{0.17em}\hspace{0.17em}\mathrm{o}\mathrm{f}\hspace{0.17em}\hspace{0.17em}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{c}\mathrm{e}\mathrm{i}\mathrm{n}\hspace{0.17em}\hspace{0.17em}\mathrm{m}\mathrm{o}\mathrm{l}\mathrm{e}\mathrm{c}\mathrm{u}\mathrm{l}\mathrm{e}\mathrm{s}}{\mathrm{l}\mathrm{i}\mathrm{p}\mathrm{o}\mathrm{s}\mathrm{o}\mathrm{m}\mathrm{e}}=\frac{I_{\left(+\right)\mathrm{T}\mathrm{r}\mathrm{i}\mathrm{t}\mathrm{o}\mathrm{n}}-I_{\left(-\right)\mathrm{T}\mathrm{r}\mathrm{i}\mathrm{t}\mathrm{o}\mathrm{n}}}{C_{\mathrm{l}\mathrm{i}\mathrm{p}\mathrm{i}\mathrm{d}}},\end{array}$ where I_(+)Triton and I_(−)Triton represent the fluorescence intensity of each sample after and before Triton X-100 treatment, respectively, and C_lipid is the total concentration of lipid.

After liver homogenization, hepatic lipids were extracted as described by Folch et al. [47 (link)]. The amount of total liver lipids was determined gravimetrically. The levels of hepatic lipids (cholesterol, triglyceride, phospholipids) and serum parameters (glucose, total cholesterol, triglyceride, non-esterified fatty acid (NEFA), total bile acids) were enzymatically determined using commercial kits (T-CHO and TG-EN; Kainos Laboratories, Tokyo, Japan; Phospholipids C-Test, Glucose CII-test, Cholesterol C-test, Triglyceride E-test, NEFA C-test and TBA-test; Wako Pure Chemical Industries, Osaka, Japan). Insulin and corticosterone were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits (Rat insulin ELISA kit; Morinaga Institute of Biological Science, Yokohama, Japan; Corticosterone ELISA kit; Assaypro, St. Charles, MO, USA).