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.
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