Chloroform methanol 2 1 solution

WBAC was measured using i-STrap (Dojindo/Dojin Glocal, Kumamoto, Japan), according to the protocol provided by the manufacturer [7 (link)]. Briefly, 100 μL of whole blood, 100 μL of saline, 10 mM of 2-diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxide (DPhPMPO), and 10 mM of tert-butyl hydroperoxide (tBuOOH) were mixed and incubated at room temperature for 30 min. In this step, tBuOOH reacted with hemoglobin in blood to produce organic radicals (tert-butyl, tert-butyloxyl, and tert-butylperoxyl radicals) through the Fenton reaction. These radicals competitively reacted with antioxidants in blood or DPhPMPO. After the incubation, DPhPMPO was extracted using 1 mL of chloroform/methanol (2:1) solution (FUJIFILM Wako) and measured by X-band ESR spectroscopy (JES-TE200; JEOL, Tokyo, Japan). The signal of DPhPMPO spin adduct intensity was corrected by marker manganese oxide intensity. When the blood contained smaller amounts of antioxidants (low blood antioxidant capacity), more radicals were trapped by DPhPMPO, and a higher ESR signal intensity was measured.

Whole-blood antioxidant capacity was measured using the i-STrap technique (Dojindo/Dojin Glocal, Kumamoto, Japan), according to the manufacturer’s protocol^{7 (link)}. Briefly, 100 μL of whole blood, 100 μL of saline, 10 mM of 2-diphenylphosphinoyl-2-methyl-3,4-dihy- dro-2H-pyrrole N-oxide (DPhPMPO), and 10 mM tert-butyl hydroperoxide (tBuOOH) were mixed and incubated at room temperature for 30 min^{7 (link)}. Then, 1 mL of chloroform/methanol (2:1) solution (FUJIFILM Wako Pure Chemical Industries, Osaka, Japan) was added and mixed for 10 min. The samples were centrifuged at 8000×g and 4 °C for 10 min, and the organic layer was collected into a new tube and stored at − 80 °C until electron spin resonance (ESR) measurement^{7 (link)}. The samples were measured by X-band ESR spectroscopy (JES-TE200; JEOL, Tokyo, Japan). The ESR conditions were as follows: microwave frequency, 9.423 GHz; microwave power, 2 mW; field center, 332.0 mT; sweep width, 20 mT; sweep time, 4 min; and time constant, 0.3 s. The signal of DPhPMPO spin adduct intensity was corrected by marker manganese oxide intensity^{7 (link)}. The number of mice in each group is shown in Supplementary Table S1.