[A new microprocedure for continuous and non-consuming determination of cellular oxygen uptake based on fluorescence...

The oxygen uptake in a cell suspension can be measured by various methods using manometric, paramagnetic or photometric techniques, oximetry, mass spectrometry or radiospectrometry. Easy-to-apply Clark-type electrochemical (polarographic) sensors are by far the most commonly used devices in medical applications. One of their drawbacks is the fact that they consume oxygen and may cause systematic errors when measuring oxygen uptake. Since the beginning of this century, concentration dependent quenching luminescence by oxygen has been used in a number of experimental settings. Using this analytical approach it is possible to detect oxygen without consuming it. We report about a new method of assessing cellular oxygen uptake using the luminescence quenching by oxygen. In an 850 microliters oxygen-tight microchamber, a fluorescent dye (tetraphenylporphyrin) adsorbed on a monolayer of gas chromatographic beads is separated from a cell suspension by a silicone membrane. An active electrochemical electrode integrated within the chamber is used to calibrate the fluorescence signal. Fluorescence is generated by green light (wavelength lambda = 546 nm), the intensity of the emitted red fluorescent light (lambda > 630 nm) is measured with a photomultiplier tube. As the first application of this new method, the oxygen uptake of human lymphocytes was determined. The cells were prepared using a routine separating technique-gradient centrifugation in Ficoll. For methodological reasons, all experiments were carried out at a temperature of 22 degrees C. In 7 consecutive measurements, an oxygen uptake of 2.81 +/- 0.85 mmol O2/10(11) lymphocytes/h was found. In less concentrated suspensions this figure is higher--an effect known as the "crowding phenomenon"--which means that with increasing cell concentration the specific oxygen uptake rate decreases. Our values for cellular oxygen uptake are higher than those in the literature. Since most reported studies on lymphocytes were done at 37 degrees C, a larger difference between our values and those in the literature would be expected. This may in part be attributed to different cell concentrations, separation techniques, etc. Another explanation might be the fact that electrochemical oxygen sensors used by other authors consume oxygen. Cell suspensions investigated with polarographic sensors therefore need to be stirred. Slow stirring reduces the values of oxygen uptake, so that a high stirring frequency is needed to obtain correct results (so-called stirring effect of polarographic electrodes). High stirring frequencies, however, destroy cells, which might be another factor explaining the results reported above. Our new method based on fluorescence quenching consumes no oxygen, and is therefore independent of stirring.