Chloroform (AR grad), sulphuric acid, methanol, acetone, iron (II

Chloroform (AR grad), sulphuric acid, methanol, acetone, iron (II) sulphate, hexane and Ringer’s solution tablets were from Merck (Darmstadt, Germany). Guanidine hydrochloride, hydrochloric acid (37%), streptomycin and C13:0 internal standard were supplied by Sigma–Aldrich Chemical (Sydney, Australia). Butylated hydroxytoluene, xylenol orange sodium salt and triphenylphosphine (99% in purify) were purchased from Alfa Aesar (Lancashire, UK). Sorbitol and hemin were bought from Sigma–Aldrich (St. Louis, USA). Sodium dithionite and KOH were purchased

from VWR Inc., (Oslo, Norway). INCB024360 cost All the other chemicals were of analytical grade as supplied. l-α-Phosphatidylcholine 95% (egg, chicken) powder (1 g) was first dissolved and mixed in 50 ml of chloroform to assure a homogeneous mixture of lipids. The organic solvent was evaporated to 1 ml by using a rotary evaporator (R215, Buchi Rotavapor, Switzerland). The solution

was dried thoroughly by nitrogen gas to a lipid residue at room temperature. Hydration of the dry lipid cake was accomplished by adding 50 ml of Ringer’s solution in a 60 °C water bath for 60 min. Liposomes were produced by using an extrusion technique, which yielded a polydisperse suspension of multilamellar liposomes. The mini-extruder was assembled by inserting two internal membrane filters and one polycarbonate membrane filter (0.1 μm pore size, Avanti polar lipids, MK2206 Inc. Alabama, USA), and then the system was heated to 60 °C before use. One gas-tight syringe (Hamilton, Bonaduz, Switzerland) was loaded with 1 ml of solution and

applied to one end of the mini-extruder while the other end of the mini-extruder was supported with an Phosphoglycerate kinase empty gas-tight syringe so that the fluid could be circulated through filters from both sides. This resulted in large, unilamellar liposome vesicles defined by the pore size of the membrane. The lipid solution was completely transferred between the original and alternative syringes by gently pushing the plunger (1 min each time) 10 times (20 passes through the membranes). A successfully prepared liposome solution had no sediment after storage at 4 °C overnight. Liposome solutions were stored at −80 °C after preparation for later use. Meat cuts were trimmed of all visible fat, frozen in liquid nitrogen and homogenised by blender (800 W Home blender, Invite) to meat powder. Hydroperoxide measurements were made on meat, with or without added liposomes. Triplicates of meat samples (0.1 g) were incubated in 1 ml of Ringer’s solution and quadruplicate meat samples were incubated in 200 μl of liposomes (4 mg/ml) and 800 μl of Ringer’s solution. To all systems, 10 μl of 20 g/l streptomycin was added and the systems were incubated for 2 h in a 37 °C water bath.

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