ESR studies of O2 uptake by Chinese hamster ovary cells during the cell cycle.

Abstract

Magnetic interactions between dissolved oxygen and nitroxide radical spin probes lead to broadening of the ESR lines. We have used a closed-chamber method based on this property to determine the maximum rate of 02 uptake per cell (Vm. per cell) in cultured mammalian cells. A suitable spin probe and a cell suspension are mixed in an aerated medium, and the rate of disappearance of dissolved 02 is measured. The effects of temperature, pH, and microwave power on the determination of dissolved oxygen in solution were studied. For Most mammalian cells are aerobic, and dissolved oxygen is vital for their growth. Elucidation of the mechanism of 02 uptake during the cell cycle is probably fundamental to the complete understanding of cell growth and cell division.There have been several reports on the measurement of 02 uptake during the cell cycle (1-4). Most previous research has been done on dividing egg cells by using the Cartesian diver technique (1) and on yeast cells by using the Clark electrode (2). To our knowledge, only two reports have appeared concerning 02 uptake by cultured mammalian cells during the cell cycle (3,4). One of the obstacles is that the existing techniques for measurement of 02 uptake by cultured mammalian cells require large amounts of.cells for each measurement, making it difficult to study mitotically synchronized cells. Most chemical means of synchronization induce multiple biochemical perturbations that obscure the interpretations of any 02 uptake measurements.It is well known that the interaction ofdissolved oxygen molecules and nitroxide free radicals through Heisenberg spin-exchange causes broadening of the ESR lines (5-7). With a suitable spin probe, this property can be used to quantitate 02 concentrations in solution (8)(9)(10).In this report, we describe an ESR closed-chamber method for determining the 02 uptake by cultured mammalian cells.Basically, a spin probe and a cell suspension are mixed in an aerated medium and the rate of disappearance of 02 is measured. Because a relatively small number of cells is required for each measurement, it has been possible in the present work to study oxygen uptake of mitotically synchronized populations of Chinese hamster ovary (CHO) cells. MATERIALS AND METHODSCell Lines. CHO cells maintained in monolayer culture from frozen stocks for several years in this laboratory were used because they are well characterized. The procedures for culturing CHO cells, either in monolayer or in suspension, and for synchronization have been described (11). The mitotic index ofcells in mitosis was about 97% and did not change appreciably during the ESR measurement.ESR Closed-Chamber Method. A mixture of 10 ml of phosphate-buffered saline (Oxoid; Dulbecco's solution A without Mg2+ and Ca2+, pH 7.4) containing 0.114 mM spin probe [3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-yloxy (CTPO); Aldrich] and 0.2% methyl cellulose E4M (Dow) or 0.1% agar (buffer A) was equilibrated with air at 370C with stirring for at least 10 min prior to use. About 5-10 X...

Saturation recovery EPR and ELDOR at W-band for spin labels

“…They introduced a useful empirical parameter to characterize line broadening, the so-called K parameter. In a study of cellular respiration, Lai et al [21] produced a careful calibration of this parameter. Hyde and Subczynski [22] realized that the superhyperfine pattern of CTPO arose not only from the methyl protons but also from the ring proton.…”

Low-field paramagnetic resonance imaging of tumor oxygenation and glycolytic activity in mice

“…The collisional interaction between TAM and dissolved paramagnetic oxygen leads to a broadening of the spectral line width of TAM. The EPR spectral broadening of TAM is linear with oxygen concentration, providing quantitative capability of EPR in determining tissue pO 2 (27). Furthermore, using magnetic field gradients, as in MRI, the spatial distribution of the TAM tracer can be obtained in a living subject (28).…”

In vivo Spectroscopy and Imaging of Nitroxide Probes

“…Alternatively, measurement of "the depth of resolution" of the NRs superhyperfine structure (e.g. parameter a/A for the CTPO shown in the Fig.1) provides similar or even better sensitivity to oxygen than perdeuterated NRs, and has been used for oxygen measurements in cellular and enzymatic systems (Backer et al, 1977;Sarna et al, 1980;Lai et al, 1982). However, both approaches still suffer from concentration-induced line broadening which is difficult to disentangle from oxygen-induced broadening.…”

“…An advantage of EPR oximetry is that it is based on pure physical interaction, Heisenberg spin exchange, between paramagnetic molecules of probe and oxygen, and does not interfere with oxygen metabolism, therefore providing a basis for noninvasive oxygen measurements in biological systems. Initially, the paramagnetic probes for EPR oximetry were almost exclusively nitroxides (Backer et al, 1977;Lai et al, 1982;Froncisz et al, 1985;Bacic et al, 1989;Chan et al, 1989;Hyde & Subszynski, 1989). Both the longitudinal (T1) and transverse (T2) relaxation times of the NRs can be affected by collisions of the nitroxide with dissolved oxygen.…”