The mixture was vortexed vigorously and then centrifuged at 24,462g for 10 min at 4 °C. The supernatant was extracted and the absorbance was measured at 407 nm against a reagent blank. Two replicates Nivolumab were measured, myoglobin solutions were used to make a linear standard curve and hemin concentrations were read from

the standard curve. Meat samples were placed into 16 × 125 mm screw-cap Pyrex culture tubes and 0.8 ml of the C13:0 internal standard, 0.56 ml of 10 N KOH in water, and 4.24 ml of MeOH were added. All tubes were incubated in a 55 °C water bath for 1.5 h with hand-shaking for 5 s every 20 min to properly permeate, dissolve and hydrolyse the samples. The samples were cooled to below room temperature and 0.464 ml of 24 N H2SO4 was added. All the tubes were incubated again in a 55 °C water bath for 1.5 h with hand-shaking for 5 s every 20 min; then the tubes were cooled again in a cold water bath and 2.4 ml of n-hexane were added to each tube. All the tubes were vortex-mixed for 5 min and centrifuged for 5 min in a table top centrifuge. The hexane layer, containing the fatty acid methyl esters, was transferred into a GC vial, capped and kept at −20 °C prior to GC analysis ( O’Fallon, Busboom, Nelson, & Gaskins, 2007). The fatty acid composition of the meat samples

was determined by gas chromatography on a fused capillary column. The oven temperature was 70 °C at the start, PR-171 solubility dmso held there for 4 min and then increased to 160 °C at a rate of 20 °C/min. Thereafter the temperature was held for a further 15 min, then the temperature was further increased at 3 °C per minute to 230 °C. Helium was used as the carrier gas at a flow rate of 68.4 ml/min at a temperature of 280 °C and the column head pressure was 309.4 kPa. Both the injector and

the detector were set at 260 °C. The split ratio was 30:1. The flame ionisation detector temperature was 290 °C with H2, air and N2 make-up gas flow rates of 40, 450 and 45 ml/min, respectively. The run time for a single sample was 92 min. C13:0 was added Hydroxychloroquine in vitro as an internal standard and used to calculate the amounts of fatty acids in muscle (mg/100 g). The fatty acids were identified by comparing their retention times with the fatty acid methyl standards. Minitab (version 16; Minitab Inc., State College PA, USA) was used for univariate regression analysis (incl. stepwise regression) and one way ANOVA. The unscrambler (version X 10.2 CAMO Software AS, Oslo, Norway) was used for principal component analysis (PCA), as well as partial least square (PLS) regression. Evaluation of the PLS regression model was with full cross-validation. Beef and chicken meat samples were incubated for different times, with or without liposomes, to examine when the largest amount of peroxides was formed. The peroxides in raw beef and chicken homogenates increased rapidly during the first 2 h of incubation at 37 °C.