Anti-oxidant and anti-atherogenic properties of liposomal glutathione: Studies in vitro, and in the atherosclerotic apolipoprotein E-deficient mice  Rosenblat Mira , Volkova Nina , Coleman Raymond , Aviram Michael   Atherosclerosis  Volume 195, Issue 2, Pages e61-e68 (December 2007) DOI: 10.1016/j.atherosclerosis.2007.05.012 Copyright © 2007 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 The effect of liposomal glutathione and control liposome on copper ion-induced LDL oxidation. LDL (100μg protein/mL) was incubated for 3h at 37°C with increasing concentrations (0–12.5μg/mL) of liposomal glutathione or control liposome (with no glutathione) and 5μmol/L CuSO4. The extent of LDL oxidation was determined by the TBARS (A) and by the lipid peroxides (B) assays. The formation of conjugated-dienes was kinetically monitored at 234nm (C). The lag time required for initiation of LDL oxidation (D) was calculated from the plots in (C). Results are given as mean±S.D. of three different experiments. Atherosclerosis 2007 195, e61-e68DOI: (10.1016/j.atherosclerosis.2007.05.012) Copyright © 2007 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 The effect of liposomal glutathione on copper ion-induced HDL oxidation. HDL (100μg protein/mL) was incubated for 3h at 37°C with increasing concentrations (0–2μg/mL) of liposomal glutathione and 5μmol/L CuSO4. (A) The formation of conjugated-dienes was kinetically monitored at 234nm. (B) The lag time required for initiation of HDL oxidation was calculated from the plots in (A). The figure shows the results of one representative experiment out of three. Atherosclerosis 2007 195, e61-e68DOI: (10.1016/j.atherosclerosis.2007.05.012) Copyright © 2007 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 The effect of liposomal glutathione consumption by E0-mice on oxidative stress in the mice peritoneal macrophages. E0-mice consumed liposomal glutathione with their drinking water (50mg/kg/day), or a similar concentration of control liposomes for 2 months. The placebo mice received only water. At the end of the study the mice peritoneal macrophages were harvested. (A) The amount of cellular total peroxides was measured by the DCFH assay. (B) Paraoxonase 2 (PON2) lactonase activity was measured using dihydrocoumarin as the substrate. Results are given as mean±S.D. of three different experiments. (*) p<0.01 vs. placebo. (#) p<0.01 vs. control liposome. Atherosclerosis 2007 195, e61-e68DOI: (10.1016/j.atherosclerosis.2007.05.012) Copyright © 2007 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 The effect of liposomal glutathione on macrophage cholesterol metabolism. E0-mice consumed liposomal glutathione with their drinking water (12.5 or 50mg/kg/day) for 2 months, or control liposomes at similar concentrations. The placebo mice received only water. At the end of the study the mice peritoneal macrophages were harvested. The cells were washed and the extent of FITC labeled lipoproteins (25μg protein/mL) uptake: native LDL (A) or Ox-LDL (B), was determined by flow cytometry. (C) After cell wash, the cells were further incubated with [3H]-acetic acid. The rate of cholesterol biosynthesis was determined by TLC as described under the Methods Section. (D) After cell wash, the extent of HDL-mediated macrophage cholesterol efflux was determined after 3h incubation. Results are given as mean±S.D. of three different experiments. (*) p<0.01 vs. placebo, (#) p<0.01 vs. control liposomes. Atherosclerosis 2007 195, e61-e68DOI: (10.1016/j.atherosclerosis.2007.05.012) Copyright © 2007 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 The effect of liposomal glutathione consumption by E0-mice on macrophage cholesterol content and on atherosclerotic lesion development. E0-mice consumed control liposome or liposomal glutathione (50mg/kg/day) with their drinking water for 2 months. At the end of the study the mice peritoneal macrophages, as well as their aortas were collected. (A) The mice peritoneal macrophages were extracted with hexane:isopropanol (3;2, v/v), and the cellular cholesterol level was determined in the dried hexane phase. (B) The atherosclerotic lesion size was measured as described in the Methods Section. Results are given as mean±S.E.M. of six mice in each group. (*) p<0.01 vs. control liposome. Atherosclerosis 2007 195, e61-e68DOI: (10.1016/j.atherosclerosis.2007.05.012) Copyright © 2007 Elsevier Ireland Ltd Terms and Conditions