Generation of Hydroxyl Radical by Enzymes, Chemicals, and Human Phagocytes In Vitro

Abstract

A B S T R A C T Methane (CH4) production from the anti-inflammatory agent, dimethyl sulfoxide (DMSO), was used to measure *OH from chemical reactions or human phagocytes. Reactions producing * OH (xanthine/xanthine oxidase or Fe++/EDTA/H202) generated CH4 from DMSO, whereas reactions yielding primarily 0r or H202 failed to produce CH4. Neutrophils (PMN), monocytes, and alveolar macrophages also produced CH4 from DMSO. Mass spectroscopy using cJ-DMSO showed formnation of d3-CH, indicating that CH4 was derived from DMSO. Methane generation by normal but not chronic granulomatous disease or heat-killed phagocytes increased after stimulation with opsonized zymosan particles or the chemical, phorbol myristate acetate. Methane production from DMSO increased as the number of stimulated PMN was increased and the kinetics of CH4 production approximated other metabolic activities of stimulated PMN. Methane production from stimulated phagocytes and DMSO was markedly decreased by purportedly potent -OH scavengers (thiourea or tryptophane) and diminished to lesser degrees by weaker -OH scavengers (mannitol, ethanol, or Indeed, stimulated neutrophils (PMN), monocytes (MN) or alveolar marcophages (AM) do produce C2H4 from methional or KMB (6-10). However, the strict dependence of C2H4 production upon -OH is in doubt (8,(10)(11)(12)(13)(14). Methional spontaneously forms C2H4, especially during prolonged incubations with tissues (8), and can also react with H202 to form C2H4 (10). The specificity of KMB as a detector of -OH is also in question because C2H4 production from KMB may reflect, at least in part, reactions with 02 or hypochlorous acid (10).In Ca++ and Mg++ (26). Contaminating erythrocytes were lysed by adding 6 ml of ice-cold sterile distilled water and mixing gently for 35 s. Tonicity was rapidly restored with 2 ml of hypertonic (4x) Ca++ and Mg++ free HBSS. The mixture was then centrifuged at 170 g for 10 min. Cells in the pellet were then washed once more, resuspended in HBSS with Ca++ and Mg++, counted, and used immediately. PMN preparations contained >98% PMN with only a few lymphocytes and rare erythrocytes, platelets or MN. Suspensions of MN were similarly prepared. The percentages of MN were determined by differential counting of 600 cells on Wright's stained smears and cytochemically confirmed by esterase stain. MN preparations contained -30% MN, <1% PMN, and the remainder lymphocytes. AM were obtained by bronchoscopic sterile saline lavage of the unaffected subsegments of the lungs of patients undergoing evaluations for localized pulmonary disease or from healthy volunteers (9). AM were recovered from lavage fluids, washed once, and counted. The final preparations contained >90% AM and <2% PMN. More than 85% of the AM excluded trypan blue. Concentrations of lymphocytes or platelets that were comparable to those remaining in phagocyte preparations did not produce significant amounts of CH4 from DMSO.Preparation of pooled human serum, opsonized zymosan, or PMA. Pooled human serum was prepared from clotted ...

Quantitation of the hydroxyl radical by reaction with dimethyl sulfoxide

“…Repine and co-workers [24] have previously reported the feasibility of using DMSO in concentrations up to 130 mM as a probe for hydroxyl radicals in human phagocytes, in experiments in which methane was measured by gas chromatography as a product of the trapping reaction, based upon the assumption that DMSO + HO  CH 3 SOOH + CH 3 was followed by CH 3 + RH  CH 4 + R. The yields of methane so produced are likely to be much greater than the yield of formaldehyde, as previously described. However, there remains the potential in some systems for competition of molecular oxygen for the methyl radicals, namely CH 3 + O 2  CH 3 OO and CH 3 OO + RH  CH 3 OOH + R, followed by reduction of the methyl hydroperoxide to methanol.…”

Scatchard analysis of methane sulfinic acid production from dimethyl sulfoxide: A method to quantify hydroxyl radical formation in physiologic systems

“…Repine and coworkers 50 have previously reported the feasibility of using DMSO in concentrations up to 130 mM as a probe for hydroxyl radicals in human phagocytes, in experiments in which methane was measured by gas chromatography as a product of the trapping reaction, based upon the assumption that DMSO + HO*  CH 3 SOOH + CH 3 * was followed by CH 3 + + RH  CH 4 + R*. The yields of methane so produced are likely to be much greater than the yield of formaldehyde, as previously described.…”

Hydroxyl radical scavengers inhibit lymphocyte mitogenesis.

“…Crude LAF preparations were prepared by incubating human PBM (5 X 106 per ml) in RPMI 1640 medium containing 5% pooled heat-inactivated human AB serum with Escherichia coli lipopolysaccharide 0111:B4 (Calbiochem) (24 ,ug/ml) for 24 10 ng/ml and does not inhibit mitogenesis up to 500 ng/ml. Me2SO, benzoate, thiourea, dimethylurea, tetramethylurea, and tryptophan, all reported to be scavengers of -OH (3,(11)(12)(13), all inhibited [3H]thymidine incorporation stimulated by PMA to a greater extent than they inhibited incorporation stimulated by Con A or PHA (Table 1). Inhibition by these agents was dose dependent and at high concentrations responses to Con A and PHA were also inhibited.…”