Determination of Alternate Pathway Complement Kinetics by Electron Spin Resonance Spectroscopy

Abstract

We describe a technic that measures the kinetics of the metabolic burst of peripheral blood neutrophils (PMN) by electron spin resonance (ESR) spin trapping. Using this technic, a functional assay for the kinetics of the alternate pathway complement cascade is developed. PMN were stimulated by phagocytosis of opsonized zymosan (OpZym) to undergo the metabolic burst and the resulting free hydroxyl radicals generated during the burst were trapped using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) to form a stable spin adduct. Rapid sequential measurements of the spin adduct concentration were made using a computer-controlled ESR spectrometer to follow the time course of the metabolic burst. Differences in the results obtained with OpZym and with PMN incubated with unopsonized zymosan (Zym) and serum yields information about the kinetics of opsonization. The kinetics of the alternate complement pathway opsonization in sera from normal and C8-deficient patients were compared and found not to be affected significantly by the absence of C8 complement protein.

Neutrophil degranulation inhibits potential hydroxyl-radical formation. Relative impact of myeloperoxidase and lactoferrin release on hydroxyl-radical production by iron-supplemented neutrophils assessed by spin-trapping techniques

“…Neutrophils [ (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) x 106/ml] were placed in 1 ml of HBSS containing ferricytochrome c, (80 ,sM), with or without cytochalasin B (5 /sg/ml), in the reference cuvette of a Perkin-Elmer model 57 spectrophotometer (Perkin-Elmer, Mountain View, CA, U.S.A.). After the addition of PMA (100 ng/ml), opsonized zymosan (3 mg/ml) or FMLP (10-5 M), absorbance was recorded continuously at 550 nm and 25 'C.…”

“…GOH has been suggested to contribute to both the microbicidal activity and the tissue damage associated with neutrophil activation in vivo [5]. Several investigators have reported detection of *OH formed by stimulated neutrophils suspended in vitro in standard buffers [6][7][8][9][10][11][12][13][14][15]. These studies implied that neutrophils possessed the endogenous capacity for GOH formation.…”

Spin Trapping

“…Although the first detection of superoxide from stimulated neutrophils was accomplished quite early, discussion raged about the role (if any) of hydroxyl radical (89) or myleopeoxidease (90) in this process. The conclusion today is that hydroxyl radical spin adduct production is highly dependent on iron (91).…”