A new method for the detection of hydroxyl radical production by phagocytic cells.

Benzoic acid, a specific scavenger of hydroxyl radical (OH.) is known to be oxidized as the result of a reaction with OH.. We have determined that the decarboxylation of benzoic acid can be used to detect OH. generated in cell-free systems and human granulocytes. Benzoic acid is oxidized by the xanthine-xanthine oxidase enzyme system. This system is known to generate O2-, H2O2 and OH.. This oxidation is inhibited by superoxide dismutase, catalase and mannitol. Therefore, the oxidation of benzoic acid occurs by a mechanism similar to that reported for the oxidation of methional to ethylene and involves OH.. Resting granulocytes do not oxidize benzoic acid. However, marked oxidation of this substrate occurs during the phagocytosis of opsonized zymosan particles, indicating the production of OH. by these cells. The reaction can be inhibited by superoxide dismutase, catalase, azide and mannitol. Therefore, the production of OH. in the cell may be similar to that observed in the cell-free system. The granulocytes of a patient with known chronic granulomatous disease did not oxidase benzoic acid, indicating a defect in the generation of OH. by these cells.

Evidence that OH. production by human PMNs is related to prostaglandin metabolism.

Recent studies indicate that human granulocytes generate OH. during the phagocytosis of zymosan particles. Several theoretical considerations suggested to us that this OH. production might be related to prostaglandin metabolism, particularly the observation that OH. is generated by the reducation of hydroperoxides in microsomal systems. In our studies, we tested the importance of prostaglandin metabolism in the production of OH. by human granulocytes (PMNs). Indomethacin and aspirin at concentrations known to impair cyclooxygenase activity decreased OH. production by PMNs during the phagocytosis of zymosan particles. Phenol, which is known to alter prostaglandin metabolism, ablated OH. completely. None of these drugs at the concentrations used impaired the generation of O-2 or H2O2 by PMNs, as indicated by their failure to diminish significantly the generation of chemiluminescence. Thus, the decrement in OH. production by these drugs could not be attributed to a nonspecific effect on the production of O-2 or H2O2. These experiments therefore, indicate that the model for OH. production observed during prostaglandin synthesis with microsomal systems applies to human granulocytes.

Trace element interactions affecting pulmonary macrophage cytotoxicity.

Effluents from the smokestacks of powerplants contain respirable particles that are enriched with a variety of biologically active trace elements. To determine the relative toxicity of trace elements in coal fly ash, the effects of selected compounds, alone and in combination, were evaluated in cultures of pulmonary alveolar macrophages. The inorganic compounds studied were ZnO, Na2SeO3, V2O5, NaAsO2, Mn3O4, and Ni3S2. Vanadium pentoxide was the most cytotoxic compound while selenium was the least toxic (approximately 100-fold less toxic than vanadium). After dose-response curves for each element were established, interactions between various compounds were studied by coexposure to trace elements at different concentrations. Selenium was completely antagonistic to the toxic effects of vanadium and slightly antagonistic to arsenic. In contrast to selenite, sulfite, a moiety with a similar redox potential, was not antagonistic to vanadium toxicity. Zinc was weakly antagonistic to the in vitro effects of nickel. No other chemical combinations displayed antagonistic, synergistic or additive effects on the function of the cultured macrophages.

Determination of hydroxyl radical production in aqueous solutions irradiated to clinically significant doses.

Decarboxylation of 14C-carboxylbenzoic acid in aqueous solutions after low-dose irradiation has been used to determine the relative magnitude of oxidation reactions and estimate the hydroxyl radicals produced. The G CO2 values determined from these measurements of 0.4 to 1.0 mM solutions of benzoic acid after x-ray doses of 1000 rads ranged from 0.72 to 0.77, in excellent agreement with values reported by authors using much higher doses of radiation. Superoxide dismutase and catalase, known scavengers of superoxide and hydrogen peroxide, respectively, did not show impairment of the oxidation of benzoic acid. On the other hand, biologically significant concentrations of phenol and mannitol appear to impair the radiation-induced oxidation of benzoic acid, indicating that the process is secondary to a reaction with OH . . We found that serum and glucose, common cell media contents, are potent OH . scavengers. These observations indicate that the oxidation of benzoic acid can be as a reliable method to estimate OH . with radiation doses of clinically significant magnitudes. In addition, these results suggest that the radiation induced by OH . in cell systems can be significantly modified by the type of buffer used.

Protein sorting by high performance liquid chromatography. 2. Separation of isophosphorylates of recombinant human DNase I on a polyethylenimine column.

Anion exchange HPLC with a polyethylenimine (PEI) column separates recombinant human deoxyribonuclease I (rhDNase) glycoforms according to the extent and positions of phosphorylation of mannose residues in N-linked oligosaccharides. The separation provides a selectivity unavailable by anion exchange HPLC with other columns or by isoelectric focusing gel electrophoresis and can be used to quantify the phosphate content of preparations of rhDNase. Tryptic mapping of fractions collected from the column and treated with alkaline phosphatase was used to identify the sites of phosphorylation. Unnatural forms of rhDNase, bearing oligosaccharide structures at only one of the two sites of glycosylation, were prepared by cleaving the phosphate-containing high mannose and hybrid structures from the purified isophosphorylates fractionated on the PEI column. The separation of rhDNase isophosphorylates on the PEI column mimics the relative affinities for the mannose 6-phosphate receptor that traffics acid hydrolases to lysosomes and provides a useful example of protein sorting by biomimetic interaction chromatography.