The purpose of this study is to examine the role of small, highly reactive molecules in the bactericidal activity of human polymorphonuclear leukocyte (PMNL), one of our most important defense against infection. This is a study that is designed to generate fundamental information as to how oxy-radicals or similar strong oxidants are generated by the PMNL. Chemical probes, introduced in small quantities coated onto latex beads, will be used to test for various oxidants that are formed in the phagosome. These probes are: 1) linoleyl alcohol because the 1,4-diene moiety is sensitive to free radical autooxidation initiated by a variety of oxy-radicals; 2) dedecyl 4-phenylbutyrate will be used to probe for the hydroxyl radical; 3) ethyl 6-(3'-5'-dimethoxyphenoxyl) hexanoate will be used to probe for and to quantify HOCl formation; and 4) ethyl 6-(phenylthio)hexanoate will be used to probe the general oxidation state of the phagosome. Products from the reaction of these compounds will be characterized by mass spectrometry. Authentic standards will be synthesized to confirm the identity of the products. Preliminary experiments have shown that iron is essential for observing peroxidation of one of the probe molecules, linoleyl alcohol. The experiments with iron gave an unusual distribution of oxidation products -- not the distribution of products that would be expected for a process initiated by the hydroxyl radical, but a distribution more like what would have been predicated if singlet oxygen were also participating in the oxidative process. Therefore, the role of iron is critical to understanding the oxidative processes in the PMNL phagosome. The individual probe molecules are designed to assess various oxidant properties and allow us to determine the nature of the oxidant that participates in bacterial killing and how this oxidant is formed. The probe molecules will be introduced to the PMNL after incorporation onto phagocytosable particles. We have been using probe molecules coated onto latex beads and we find that this delivery system activates the PMNLs and causes them to under go all of the processes normally associated with bacterial killing. The use of latex particles coated with probe molecules in a buffer containing only glucose eliminates many of the potential ambiguities associated with more complex systems.
