Hydrogen Sulfide and its Potential Role as an Oxygen Sensor

Hydrogen sulfide has in the last 15 years been elevated from its previous place as nothing more than a toxic gas to the status of ubiquitous gasotransmitter with numerous physiologic functions. In this thesis the focus is two-fold: whether H2S functions as a gasotransmitter in blood and whether H2S could act more locally as an endogenous oxygen sensor. The initial research presented here examines sulfide measurement techniques for biologic samples, and assessed their applicability for use in blood and plasma. It also shows the development of an amperometric means of conducting this research. It was found that the standard protocol for potentiometric measurement of sulfide in plasma is unsuitable for such use. The experimental conditions liberate sulfide from cysteines in plasma proteins, creating artificially high sulfide measurements. Using the methylene blue method we were unable to replicate blood sulfide measurements found in other studies, while showing unequivocally that this method should work in plasma if sulfide is present. The amperometric sensor developed here may be used in real time without altering the sample. Using this method we showed in blood from numerous animals that free H2S is undetectable in blood and that blood consumes sulfide quite rapidly. The second portion of this research showed that tissues consume endogenously-produced H2S oxygen-dependently and that the likely location of this consumption is in the mitochondria. The inhibitory level of oxygen was quite low, and the levels of cysteine required to stimulate measurable production were supraphysiologic, thus an attempt was made to develop a means of measuring intracellular sulfide. This was done through sulfide-sensitive Photonic Explorers for Bioanalysis with Biologically Localized Embedding (PEBBLE). PEBBLEs were produced in an organically-modified silica matrix using the sulfide-sensitive fluorescent dye fluorescein mercuric acetate. While the PEBBLEs were sensitive to H2S and insensitive to cysteine, intracellular production of sulfide was not successfully demonstrated. H2S may function as an oxygen sensor intracellularly, but it does not circulate in the blood and is unlikely to exist outside of localized pockets within cells due to rapid oxidation. Further work should be done to establish a means of intracellular measurement of H2S.