The samples were mixed with 1 ml of chloroform and methanol (2:1, volume-ratio), vortexed and centrifuged at 24,462g for 10 min at 4 °C. After centrifugation the system separated into three phases which were 1.33 ml of polar upper phase (25% methanol + 75% Ringer’s solution, pH 7), an interphase (the meat protein aggregate) and 0.67 ml of non-polar lower Roxadustat phase (chloroform) containing soluble lipids. Each of the three phases was removed for separate hydroperoxide measurements. Upper phase (700 μl) was removed and the following chemicals were added immediately in this order: 5 μl of 4 mM BHT, 4 μl of 2 M
H2SO4, 40 μl of H2SO4 at pH 1.8, 30 μl of 5 mM XO + 5 M sorbitol mixture at pH 1.8 and 40 μl of 1.67 mM FeSO4 at pH 1.8. A blank containing the upper phase reduced with 10 μl of 1 M sodium dithionite and subjected to an identical protocol was used as a negative control. The protein aggregate at the interphase was washed three times with 2:1 chloroform:methanol before 1.7 ml of 6 M GuHCl were added to resolubilise the protein for optimal hydroperoxide exposure. The protein aggregate did not always solubilise to a transparent solution, but it swelled to an open system that allowed for low molecular
weight diffusion (i.e. diffusion of the chemicals added). After 30 min of solubilisation, all chemicals were added immediately in this order: 12 μl of 4 mM BHT, 97 μl of H2SO4 at pH 1.8, 73 μl of 5 mM XO + 5 M sorbitol mixture at pH 1.8 and 73 μl of 1.67 mM FeSO4 at pH 1.8. A blank containing suspended protein phase reduced with 10 μl of 1 M sodium dithionite and subjected to identical protocol was used as a negative control. Lower phase (50 μl CH5424802 chemical structure chloroform) was removed and chemicals were added immediately in this order: 200 μl of chloroform, 460 μl of methanol, 5 μl of 4 mM BHT, 12 μl of 2 M H2SO4, 26 μl of 10 mM XO at pH 1.8 and 54 μl of 1.67 mM FeSO4 at pH 1.8. A blank containing the lower phase reduced with 10 μl of 1 M triphenylphosphine
and subjected to identical protocol was used as a negative control. All the samples were incubated for 60 min in enclosed Eppendorf tubes at room temperature to ensure colour development. The upper phases and the suspended Thalidomide protein interphases were centrifuged at 24,462g for a further 10 min at 4 °C to secure transparency before the measurements by the spectrophotometer, while the lower phases were measured spectrophotometrically at 590 nm immediately after the incubation. The initially obtained hydroperoxide values were calculated by first subtracting the negative control, then the absorbance was divided by the pigments’ molar absorptivities of 14,840 (1 cm pathway) and 87,583 (1 cm pathway) for the upper phase/inter phase and the lower phase, respectively, before correcting for dilution. Our procedure is a modification of Gay and Gebicki (2002a), but adapted to meat instead of serum and with reduced volumes to adapt the technique to Eppendorf tubes.