Reverse-phase HPLC fractionation and identification of low-molecular–weight components in plasma used by MPO to initiate lipid peroxidation.

Plasma was filtered through a 10-kd MWt cutoff filter. The filtrate of plasma containing the low-molecular–weight components was then fractionated by reverse-phase HPLC as described in “Materials and methods.” Each column fraction was dried under N₂, reconstituted in 50 mM sodium phosphate buffer (pH 7.0), and incubated with a lipid source (LDL, 0.2 mg/mL), isolated human MPO (30 nM), and an H₂O₂-generating system (G/GO) (10 μM/h flux of H₂O₂). After incubation at 37°C for 12 hours, the contents of 9-HETE, 9-HODE, and CE-HODEs were then determined as described in “Materials and methods.” Retention times of some compounds described as MPO substrates in vitro include: Cl⁻ and Br⁻, fraction 2 (F2); SCN⁻, F3; NO$2−$⁠, F4; ascorbic acid, F5; tyrosine, F8; 6-hydroxy-dopamine, F19; serotonin, F20; catecholamines, F18-23; estradiols, F26.