Abstract
Small uni-lamellar vesicles were prepared from 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) with 1% spin label of 1-palmitoyl-2-(16-doxylstearoyl)phosphatidylcholine (16-PC). POPC membranes are in the fluid phase in the physiological temperature range, which ensures high solubility of oxygen in the membrane center where the 16-PC nitroxide moiety is located. The nitroxide moiety is isolated from cellular reductants and paramagnetic ions that might interfere with spin-label oximetry measurements. This analyte is stable in cell suspensions and can be used in studies as long as 400 min. The saturation recovery EPR approach, possibly for the first time, was used to study cellular respiration of rat dopaminergic neuronal cells. It is widely accepted that this approach is the most direct way to carry out oximetric studies. The preliminary experiments were carried out at X-band for closed chamber geometry. Oximetric measurements were performed for cell concentrations from 750 to 5000 cells in one µL of culture media. Results confirmed that the oxygen consumption rate was linearly proportional to the number of cells. Furthermore at the lower cell concentration, we found a breaking point in the rate which is not due to diffusion limitation but is due to the change in cell metabolism because there is not enough oxygen, consistent with Michaelis-Menten kinetics. In the developed analyte, the same hydrocarbon environment, which dissolves oxygen very well, always surrounds nitroxide moieties of spin-label molecules. Therefore, the partial pressure of oxygen influence the EPR spectrum of spin-labels in the analyte. Such microscopic analytes are readily and uniformly distributed within the sample, thus giving a rapid response to changes in oxygen partial pressure. The improved oximetry methodology developed here presents a high impact opportunity for non-invasive assessment of disease mechanisms and response to therapeutic intervention.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
