OxyFile #103

TI:  H2O2 Release from Human Granulocytes during Phagocytosis
     I. Documentation, Quantitation, and Some Regulating Factors

DT:  September 17, 1974

AU:  R.K Root, J. Metcalf, N. Oshino, B. Chance

SO:  J. Clin. Invest, Vol. 55, May 1975, 945-955

AB:  The extinction of fluorescence of scopoletin during its 
     oxidation by horseradish peroxidase (HPO) provides a highly 
     sensitive and specific assay for small quantities of 
     peroxide in solution.  With this assay, the release of free 
     H2O2 into the extracellular medium by phagocytizing human 
     granulocytes has been documented and quantitated, and some 
     of the regulating factors have been determined.  Under basal 
     conditions granulocytes released less than 0.01 nmol/ml of 
     H2O2 (2.5 x 10(6) polymorphonuclear leukocytes/ml).  Upon 
     the addition of phagocyte particles (latex, opsonized yeast, 
     or staphylococci), an abrupt increase in extracellular 
     peroxide concentration was observed (>50-fold above basal 
     levels) after latencies as short as 10 s.  Release reflected 
     increased intracellular H2O2 production during phagocytosis 
     in that it paralleled the respiratory burst and was absent 
     when phagocytosis was prevented or when cells from patients 
     with chronic granulomatous disease were utilized.  Evidence 
     that scopoletin oxidation occurred predominantly in the 
     extracellular medium was obtained by demonstrating a marked 
     inhibition when HPO was omitted from the reaction mixture or 
     when exogenous catalase was added.  Similarly, it was found 
     that exogenous serum also inhibited scopoletin oxidation, 
     apparently because of the presence of competing hydrogen 
     donors.

     H2O2 formation and release were observed at rates which 
     closely paralleled those of phagocytosis.  With O2 
     consumption as an approximate index of H2O2 formation, the 
     fractions released during maximal rates of particle uptake 
     were calculated as follows: for latex, 15.7%; for 
     staphylococci, 10.3%; and for yeast, 4.9%.  It is postulated 
     that release is due to diffusion of free H2O2 from an 
     expanded intracellular pool of this substance that develops 
     during phagocytosis.  This pool represents the net of 
     increased synthesis versus catabolism by various enzymatic 
     pathways for H2O2 disposal within the cells.

     The close relationship between rates of H2O2 formation and 
     rates of phagocytosis by human granulocytes suggest a role 
     for specialized areas of the cell membrane, involved in 
     particle ingestion, in the trigger mechanism for H2O2 
     synthesis.  The consequences of H2O2 release to other cells 
     or organisms in the immediate environment of phagocytizing 
     granulocytes remain to be determined.