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...

A nitric oxide (•NO) spin‐trapping technique combined with electron paramagnetic resonance (EPR) spectroscopy has been employed to measure the in vivo production of •NO in lipopolysaccharide (LPS)‐treated mice. The in vivo spin‐trapping of •NO was performed by injecting into mice a metal—chelator complex, consisting of N‐methyl‐d‐glucamine dithiocarbamate (MGD) and reduced iron (Fe2+), that binds to •NO and forms a stable, water‐soluble [(MGD)2‐Fe2+‐NO] complex, and by monitoring continuously the in vivo formation of the latter complex using an S‐band EPR spectrometer. At 6 h after intravenous injection of LPS, a three‐line EPR spectrum of the [(MGD)2‐Fe2+‐NO] complex, was observed in the blood circulation of the mouse tail; the [(MGD)2‐Fe2+] complex was injected subcutaneously 2 h before EPR measurement. No signal was detected in control groups. Administration of N G‐monomethyl‐l‐arginine, an •NO synthase inhibitor, caused a marked reduction in the in vivo EPR signal of the [(MGD)2‐Fe2+‐NO] complex, suggesting that the •NO detected is synthesized via the arginine‐nitric oxide synthase pathway. The results presented here demonstrated, for the first time, the in vivo real time measurement of •NO in the blood circulation of conscious, LPS‐treated animals.

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An unusual ESR display has been developed that exhibits sensitivity to bimolecular collisions of dissolved oxygen in water with nitroxide radical spin probes at oxygen concentrations as low as 0.1 guM, requiring only 1 dul of sample. The method involves observation of the ESR rapid-passage signal when tuned to the dispersion using a loop-gap resonator. The bimolecular collision rate determines the phase of the signal. The method has been used in a closed-chamber geometry to study respiration of asynchronous populations of Chinese hamster ovary (CHO) cells. An integral of the Michaelis-Menten equation permits direct comparison with experiment and is shown to be incompatible with the data. The theory of diffusion limitation also is developed and shown to be inconsistent with experiment. The average oxygen concentration is found to decrease as V1,,t, where t is the time after sealing the chamber, to a critical oxygen concentration of 5.2 juM. Below 5.2 IAM, the concentration can be fitted to an exponential form, exp(-t/T), where X = 15 sec for 4000 cells per Il. It is believed that this experimental behavior is determined by complex enzyme kinetics.In a series of recent papers (1-7), we have developed the methodology of spin-label oximetry and have applied it to various cellular and liposomal membrane systems. The physical basis of the method depends on Heisenberg exchange between nitroxide radical spin labels and molecular oxygen, which occurs at the bimolecular collision rate. This rate can be estimated by using the Smoluchowski equation,where R is the interaction distance, which is generally assumed to be 4.5 A, and D is the diffusion constant of 02, which is assumed to be much greater than the diffusion constant of the spin label. These exchange events shorten both the spin-lattice relaxation time (T1) and the spin-spin relaxation time (T2) of the spin label.The T2 oximetric method was introduced by Backer et al. Heisenberg exchange between a fast-relaxing species (e.g., molecular oxygen) and a slow-relaxing species (e.g., a spin label) provides a coupling of the spin label to the lattice, as pointed out by Hyde and Sarna (10). This is the basis of the spin-label T1 method (1, 2, 5). One advantage of this method is that the spin label can be undergoing slow motion with no proton structure evident.One of the rationales for spin-label oximetry is that a molecular probe is used. It is a simple matter to vary the concentration of the probe to verify that it does not itself interfere with the kinetics of oxygen uptake (3). Polarographic probes are known to distort the distribution of oxygen in the sample under investigation (11, 12). Another rationale is that both T1 and T2 of spin labels happen to be rather close to the bimolecular collision rate of oxygen dissolved in water under standard conditions, which gives the method good sensitivity. Fluorescence probes are sometimes used for oximetry, where quenching of fluorescence by oxygen is the observed process, but there is a poor match between collision rate...

We describe here a spin-trapping method combined with X-band electron paramagnetic resonance (EPR) spectroscopy for ex vivo measurement of nitric oxide (.NO) levels in the urine of both normal and lipopolysaccharide (LPS)-induced shock mice. Normal or LPS-treated mice were injected subcutaneously with a metal-chelator complex, N-methyl-D-glucamine dithiocarbamate-ferrous iron, [(MGD)2/Fe], which binds to .NO and forms a water-soluble [(MGD)2/Fe-NO] complex. At 2 h after injection of the [(MGD)2/Fe] complex, a three-line EPR signal characteristic of the [(MGD)2/Fe-NO] complex was detected in the urine of either normal or LPS-treated mice. It is estimated that the concentrations of the [(MGD)2/Fe-NO] complex in normal and LPS-treated mouse urine were 1.3 and 35 microM, respectively. This 25-fold increase in .NO levels in the LPS-treated mouse urine provides the direct evidence that LPS challenge induces the overproduction of .NO in mice. Administration of N-monomethyl-L-arginine (NMMA; 50 mg/kg) inhibited the ex vivo signal intensities of the [(MGD)2/Fe-NO] complex in the urine of either normal or LPS-treated mouse urine. Furthermore, after injection of 15N-arginine (10 mg per mouse), a composite EPR spectrum, consisting of a three-line spectrum of the [(MGD)2/Fe-14NO] complex and a two-line spectrum of the [(MGD)2/Fe-15NO] complex, was detected in the urine. These isotopic tracer experiments further confirm that the detected .NO levels in the mouse urine are produced via the arginine-nitric oxide pathway. This ex vivo spin-trapping method should readily be adapted to experiments on larger animals and provide a noninvasive way of measuring both constitutive and inducible .NO synthase activities in living animals under physiological as well as pathophysiological conditions where .NO is overproduced.