J. Phys. Chem. B 2002, 106, 11929-11935
11929
Electrochemical Preparation and EPR Studies of Lithium Phthalocyanine. 4. Effect of Nitric Oxide Govindasamy Ilangovan, Ranjeeta Pal, Jay L. Zweier, and Periannan Kuppusamy* Biomedical EPR Imaging Center, Dorothy M. DaVis Heart & Lung Research Institute, The Ohio State UniVersity, 420 West 12th AVenue, Columbus, Ohio 43210 ReceiVed: June 21, 2002; In Final Form: August 21, 2002
The suitability of microcrystalline particles of lithium phthalocyanine (LiPc) as probes for electron paramagnetic resonance (EPR)-based determination of the concentration of molecular oxygen (O2) in biological systems was previously investigated. In the present study, the interaction of nitric oxide (NO) with LiPc and the resultant characteristics of EPR line shape were investigated. The line width was linearly dependent upon the partial pressure of NO (pNO) in the range 0-100 mmHg, whereas beyond 100 mmHg, the relationship was nonlinear. A line-broadening mechanism on the basis of a dual spin model, similar to that proposed for the magnetic interaction of molecular oxygen (O2) with LiPc, is used to interpret the linear dependence. The saturation behavior observed at higher pNO is interpreted on the basis of saturation in the surface coverage due to NO adsorption. The adsorption equilibrium was analyzed utilizing a model similar to the Langmuir adsorption isotherm. During dynamic equilibrium at ambient pressure and under 10% NO concentration, more than 85% surface coverage was apparent. The adsorption/desorption rates of NO were also determined by this model. It is concluded from this study that the line broadening of the EPR spectrum of LiPc occurs due to adsorption of NO and dipolar interaction rather than spin exchange. This behavior is similar to that of LiPc upon interaction with oxygen. Long-term exposure of LiPc to NO showed irreversible adsorption, resulting in a second broad peak, which was insensitive to further exposure to NO. However, irreversible adsorption of O2 predominates over NO in vivo. Nitric oxide synthase (NOS) inhibitor (L-NAME) or NO donor (sodium nitroprusside) did not affect the magnitude of adsorption of O2. These results clearly show that the EPR oximetry method that utilizes LiPc probe is not sensitive enough to determine the in vivo levels of NO (110 nM) in the presence of physiological concentrations of O2.
Introduction Our recent studies have focused on the preparation, characterization, and use of a microcrystalline powder of lithium phthalocyanine (LiPc) as a highly sensitive probe for lowfrequency electron paramagnetic resonance (EPR) oximetry for the determination of concentration of molecular oxygen (O2) in biological systems.1-4 We observed earlier that the magnetic interaction between the neutral radicals of LiPc and paramagnetic O2 molecules was intriguing and highly complex. Our results agree with several recent reports, which describe various fundamental characteristics of LiPc such as spin dynamics, magnetic moment exchange, surface diffusion, particle sizedependent line width, O2 sensitivity, and adsorption.5-10 We recently reported the use of a microcrystalline powder of LiPc for simultaneous determination of O2 and superoxide (O2•-) produced during in vitro enzymatic and cellular reactions in a 50 µL reaction mixture.3 The present study describes the interaction of nitric oxide (NO) with LiPc and the suitability of this material as a probe for determination of NO. The LiPcbased detection of NO is analogous to EPR oximetry, which is based on the principle of Heisenberg magnetic exchange interaction with paramagnetic molecules and the spin probe. As NO is paramagnetic with an unpaired electron, it can also * Address for correspondence: Periannan Kuppusamy, Ph.D., Biomedical EPR Imaging Center, The Ohio State University, 420 West 12th Ave., TMRF-114, Columbus, OH 43210, E-mail:
[email protected]. Phone: 614-292-8998. Fax: 614-292-8454.
induce spin relaxation of LiPc upon contact, a phenomenon that can be taken as an advantage in the quantitative determination of NO, as in the case of O2. The induced relaxation increases linearly as the concentration of these paramagnetic molecules increases, with a concomitant increase in the frequency of collision.11 However, in the case of solid spin probes, the linear relationship between the concentration of paramagnetic molecules and line width does not hold well due to various factors such as adsorption, surface diffusion, different energetics of the spin states, etc. The underlying mechanism of O2- or NOinduced EPR line broadening of LiPc is entirely different due to the self-interacting nature of the LiPc spins.2,12 We recently observed that the adsorption of the O2 plays a major role.13 The adsorption of O2 onto the paramagnetic probes (surface or bulk) is simple and identified as physisorption. The adsorption of NO, on the other hand, is more complex, as observed by several investigators.10,14-19 The adsorption of NO onto metal and metal oxide has been thoroughly studied with an emphasis on the catalytic decomposition of NO. Unlike O2, NO is known to form strong coordination complexes with the transition metal ions (TMI) of the host matrix,14-16 leading to marked alterations in the characteristics of the EPR spectra of the host metal ions. FTIR analysis of NO-adsorbed metal oxides has yielded characteristic bands in the FTIR spectrum that correspond to cationic (TMI-NO+) and anionic (TMI-NO-) nitrosyl complexes.14,16,18,19 EPR spectroscopy has been used as a suitable technique to study the nature of the NO coordination with host
10.1021/jp026360l CCC: $22.00 © 2002 American Chemical Society Published on Web 10/24/2002
11930 J. Phys. Chem. B, Vol. 106, No. 46, 2002 metal ions, as such coordination drastically affects the native TMI.15 However, there are no reports so far on the adsorption of NO onto a paramagnetic crystal without TMI and its magnetic interaction. Therefore, in the present study we have investigated the adsorption of NO onto the microcrystals of LiPc and the resultant magnetic interactions. Our current results revealed that the adsorption of NO onto the microcrystals of LiPc did not affect the nature of the original EPR spectrum of LiPc due to involvement of any chemical bonding but resulted in a spin exchange between the adsorbed NO and the LiPc, leading to marked changes of greater magnitude in the EPR spectral line width. Experimental Details LiPc Synthesis. LiPc was synthesized by the constant potential electrolysis method, as described in our previous report.1 The synthesized material was characterized for its purity and crystalline nature by optical microscopy and X-ray diffractions. Micrographs revealed the formation of needle-shaped crystals. XRD and EPR data showed that the product was exclusively in x form as microcrystalline powder. EPR Measurements. EPR measurements were carried out at the X-band, 9.82 GHz, using a Bruker ER 300 spectrometer with a TM110 microwave cavity. Data acquisition and analysis were performed using a personal computer interfaced to the spectrometer. Instrument control, data acquisition, and processing were performed using SPEX, Personal Computer software developed in our laboratory. Because the peak-to-peak width of the LiPc spectrum was extremely small (anoxic width
