Early Observations In Free Radical Pathology – Early History
Majid Ali, M.D.
An Article of Course on Molecular Biology of Oxygen
In 1983, Eli Metchnikoff, a Russian biologist, made his seminal discovery of the role of phagocytes in host defense. His work provided the scientific basis for our current ideas of cellular host defense. During the five decades which followed Metchnikoff’s work, the investigative focus remained on the morphologic cellular and subcellular changes.
The next major advance in our understanding of the phagocytic function came from the work of Baldridge and Gerard. These investigators in 1933 demonstrated the essential changes in the oxygen generation and oxygen consumption during cellular host defense. They reported increased oxygen uptake by canine neutrophils during phagocytosis of Sarcin lutea. Increased oxygen uptake was thought to be related to increased oxygen consumption and higher energy needs during phagocytosis. The true significance of this observation, however, was not recognized for the next 25 years.
Karnovsky in 1959 elucidated the phenomenon of increased oxygen uptake and linked it to energy production rather than oxygen consumption related to ATP formation. He observed that increase in oxygen uptake associated with phagocytosis could not be blocked by cyanide, and thus could not be related to oxygen consumption. Evidently, oxygen was being used by the phagocyte for some other function.
Hydrogen Peroxide Production
The next major advance in our understanding of the biochemical events occurring within the phagocyte came from the work of Quastel in 1965. He demonstrated that the phagocyte during the phenomenon of phagocytosis liberates H2O2 into its environment. Quastel further proposed that the phagocyte generates H2O2 as an antimicrobial agent. What was the reducing equivalent in the biochemical equation of H2O2 production within each phagocyte? Since it was known at that time that the rate of glucose oxidation via the hexose monophosphate shunt is increased in the phagocyte engaged in phagocytosis, it seemed reasonable to assume that the reducing equivalents for H2O2 production were provided by this shunt.
The metabolic event leading to the increased oxygen uptake and generation of H2O2 then could be expressed as follows:
Reduced pyridine nucleotide + O2
—-> oxidized pyridine nucleotide + H2O2
This sequence of events came to be known as RESPIRATORY BURST.
The relevance of these essential biochemical events occurring at the cellular level was demonstrated by the landmark work of Good and his colleagues in 1957. These investigators described the clinical entity of chronic granulomatous disease (CGD) which is characterized by recurrent bacterial and fungal infections. Very low levels of oxidase activity are found in the neutrophils of this disease. This line of evidence supports the hypothesis (originally put forth by Quastel) that the increased susceptibility to infections in CGD is due to the failure of the phagocytes to bring about the intracellular biochemical events designated as respiratory burst.
Hydrogen peroxide has a weak microbicidal activity. It was recognized by these early workers that this molecule by itself cannot provide an effective defense against the microorganism. It seemed highly likely that hydrogen peroxide served as a precursor for degeneration of other free radicals with more potent antimicrobial activity. The first line of evidence to support such hypothesis came from the work of Kelbanoff concerning the role of myeloperoxidase in oxygen-dependant killing of microorganisms with phagocytes. In 1967, Klebanoff showed the antimicrobial effectiveness of the halide ion. Further work along these lines showed that myeloperoxidase catalyzes the oxidation of C1– by hydrogen peroxide to produce HOC1 which has very potent antimicrobial propertie